Antibodies that bind novel pcsk9 variants

ABSTRACT

The present invention provides novel polynucleotides encoding PCSK9b and PCSK9c polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel PCSK9b and PCSK9c polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application is a continuation application of U.S. Ser. No.14/158,063, filed Jan. 17, 2014, which is a continuation application ofU.S. Ser. No. 13/720,000, filed Dec. 19, 2012, now abandoned, which is adivisional application of U.S. Ser. No. 13/336,371, filed Dec. 23, 2011,now granted U.S. Pat. No. 8,354,264, which is a divisional applicationof U.S. Ser. No. 12/903,658, filed Oct. 13, 2010, now granted U.S. Pat.No. 8,105,804, which is a divisional application of U.S. Ser. No.12/456,798, filed Jun. 23, 2009, now granted Pat. No. 7,846,706, whichis a divisional application of U.S. Ser. No. 11/824,461, filed Jun. 28,2007, now granted U.S. Pat. No. 7,572,618, which claims the benefit ofprovisional application U.S. Ser. No. 60/818,234 filed Jun. 30, 2006.The entire teachings of the referenced application are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding PCSK9b andPCSK9c polypeptides, fragments and homologues thereof. Also provided arevectors, host cells, antibodies, and recombinant and synthetic methodsfor producing said polypeptides. The invention further relates todiagnostic and therapeutic methods for applying these novel PCSK9b andPCSK9c polypeptides to the diagnosis, treatment, and/or prevention ofvarious diseases and/or disorders related to these polypeptides. Theinvention further relates to screening methods for identifying agonistsand antagonists of the polynucleotides and polypeptides of the presentinvention.

BACKGROUND OF THE INVENTION

Atherosclerosis is a disease of the arteries responsible for coronaryheart disease (CVD) that underlies most deaths in industrializedcountries (Lusis, 2000). Several risk factors for CHD have now been wellestablished: dyslipidemias, hypertension, diabetes, smoking, poor diet,inactivity and stress. The most clinically relevant and commondyslipidemias are characterized by an increase in beta-lipoproteins(VLDL and LDL particles) with hypercholesterolemia in the absence orpresence of hypertriglyceridemia (Fredrickson et al, 1967). An isolatedelevation of LDL cholesterol is one of the most common risk factors forCVD. Twin studies (Austin et al, 1987) and family data (Perusse, 1989;Rice et al, 1991) have shown the importance of genetic factors in thedevelopment of the disease, particularly when its complications occurearly in life. Mendelian forms of hypercholesterolemia have beenidentified: at first the autosomal dominant form (ADH) (Khachadurian,1964) and later the autosomal recessive form (ARH), initially describedas “pseudohomozygous type II hyperlipoproteinemia” (Morganroth et al,1967).

ADH is a heterogeneous genetic disorder. Its most frequent andarchetypal form is Familial Hypercholesterolemia (FH) with a frequencyof 1 in 500 for heterozygotes and 1 per million for homozygotes(Goldstein et al, 1973). The disease is co-dominant with homozygotesbeing affected earlier and more severely than heterozygotes. FH iscaused by mutations in the gene that encodes the LDL receptor (Goldstein& Brown, 1978) (LDLR at 19p13.1-p13.3) (MIN 143890). It is characterizedby a selective increase of LDL cholesterol levels in plasma giving riseto tendon and skin xanthomas, arcus corneae and cardiovascular depositsleading to progressive and premature atherosclerosis, CHD and mortality(occurring before 55 years). The second form of ADH is FamilialDefective apo B-100 (FDB) caused by mutations in the apolipoprotein Bgene (APOB at 2p23-p24), encoding the ligand of the LDL receptor(Inneraty et al, 1987) (MIN 144010). The existence of a greater level ofgenetic heterogeneity in ADH (Saint-Jore et al, 2000) has been reportedand the implication of a third locus named HCHOLA3 (formerly FH3) hasbeen detected and mapped at 1p34.1-p32 in a French family (Varret et al,1999) (MIM 603776). These results were confirmed by Hunt et al. in alarge Utah kindred (Hunt et al, 2000).

PCSK9, for Eroprotein Convertase Subtilisin/Kexin type 2 (also referredto as HCHOLA3, NARC-1, or FH3) is a protease belonging to the proteinaseK subfamily of the secretory subtilase family (Naureckiene et al., Arch.of Biochem. And Biophy., 420:55-57 (2003)). PCSK9 has been shown to playa role in cholesterol homeostasis by regulating apolipoprotein receptorsecretion. It may also have a role in the differentiation of braincortical neurons (Seidah et al., PNAS 100(3):928-933 (2003)).

The wild type PCSK9 gene contains 12 exons. The translated proteincontains a signal peptide in the NH2-terminus, and in cells and tissuesan about 74 kDa zymogen (precursor) form of the full-length protein isfound in the endoplasmic reticulum. During initial processing in thecell, the about 14 kDa prodomain peptide is autocatalytically cleaved toyield a mature about 60 kDa protein containing the catalytic domain anda C-terminal domain often referred to as the cysteine-histidine richdomain (CHRD) (FIG. 1). This about 60 kDa form of PCSK9 is secreted fromliver cells. The secreted form of PCSK9 appears to be thephysiologically active species, although an intracellular functionalrole of the about 60 kDa form has not been ruled out.

Several mutant forms of PCSK9 are known, including S127R, N157K, F216L,R218S, and D374Y, with S127R, F216L, and D374Y being linked to autosomaldominant hypercholesterolemia (ADH). Benjannet et al. (J. Biol. Chem.,279(47):48865-48875 (2004)) demonstrated that the S127R and D374Ymutations result in a significant decrease in the level of pro-PCSK9processed in the ER to form the active secreted zymogen. As aconsequence it is believed that wild-type PCSK9 increases the turnoverrate of the LDL receptor causing inhibition of LDL clearance (Maxwell etal., PNAS, 102(6):2069-2074 (2005); Benjannet et al., and Lalanne etal), while PCSK9 autosomal dominant mutations result in increased levelsof LDLR, increased clearance of circulating LDL, and a correspondingdecrease in plasma cholesterol levels (Rashid et al., PNAS,102(15):5374-5379 (2005).

Lalanne et al. demonstrated that LDL catabolism was impaired andapolipoprotein B-containing lipoprotein synthesis was enhanced in twopatients harboring S127R mutations in PCSK9 (J. Lipid Research,46:1312-1319 (2005)). Sun et al. also provided evidence that mutantforms of PCSK9 are also the cause of unusually severe dominanthypercholesterolaemia as a consequence of its affect of increasingapolipoprotein B secretion (Sun et al, Hum. Mol. Genet., 14(9):1161-1169(2005)). These results were consistent with earlier results whichdemonstrated adenovirus-mediated overexpression of PCSK9 in mice resultsin severe hypercholesteromia due to durastic decreases in the amount ofLDL receptor Dubuc et al., Thromb. Vasc. Biol., 24:1454-1459 (2004), inaddition to results demonstrating mutant forms of PCSK9 also reduce thelevel of LDL receptor (Park et al., J. Biol. Chem., 279:50630-50638(2004). The overexpression of PCSK9 in cell lines, includingliver-derived cells, and in livers of mice in vivo, results in apronounced reduction in LDLR protein levels and LDLR functional activitywithout changes in LDLR mRNA level (Maxwell K. N., Breslow J. L., Proc.Nat. Amer. Sci., 101:7100-7105 (2004); Benjannet S. et al., J. Bio.Chem. 279: 48865-48875 (2004)).

Using the above examples, it is clear the availability of novel forms ofPCSK9 provide an opportunity for the identification of PCSK9 agonists,as well as, in the identification of PCSK9 inhibitors. All of whichmight be therapeutically useful under different circumstances.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of PCSK9b and PCSK9c polypeptides or peptides usingrecombinant techniques. Synthetic methods for producing the polypeptidesand polynucleotides of the present invention are provided. Also providedare diagnostic methods for detecting diseases, disorders, and/orconditions related to the PCSK9b and PCSK9c polypeptides andpolynucleotides, and therapeutic methods for treating such diseases,disorders, and/or conditions. The invention further relates to screeningmethods for identifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding thePCSK9b protein having the amino acid sequence shown in FIGS. 1A-C (SEQID NO:2), respectively, or the amino acid sequence encoded by the cDNAclone, PCSK9b (also referred to as PCSK9-b), deposited as ATCC® DepositNumber PTA-7622 on May 10^(th), 2006.

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding thePCSK9c protein having the amino acid sequence shown in FIGS. 1A-D (SEQID NO:4), respectively, or the amino acid sequence encoded by the cDNAclone, PCSK9c (also referred to as PCSK9-c), deposited as ATCC® DepositNumber PTA-7622 on May 10^(th), 2006.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of PCSK9b or PCSK9c polypeptides or peptides usingrecombinant techniques. Synthetic methods for producing the polypeptidesand polynucleotides of the present invention are provided. Also providedare diagnostic methods for detecting diseases, disorders, and/orconditions related to the PCSK9b or PCSK9c polypeptides andpolynucleotides, and therapeutic methods for treating such diseases,disorders, and/or conditions. The invention further relates to screeningmethods for identifying binding partners of the polypeptides.

The invention further provides an isolated PCSK9b or PCSK9c polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

The invention further relates to a polynucleotide encoding a polypeptidefragment of SEQ ID NO:2 or SEQ ID NO:4, or a polypeptide fragmentencoded by the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to a polynucleotide encoding a polypeptidedomain of SEQ ID NO:2 or SEQ ID NO:4 or a polypeptide domain encoded bythe cDNA sequence included in the deposited clone, which is hybridizableto SEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to a polynucleotide encoding a polypeptideepitope of SEQ ID NO:2 or SEQ ID NO:4 or a polypeptide epitope encodedby the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to a polynucleotide encoding a polypeptideof SEQ ID NO:2 or SEQ ID NO:4 or the cDNA sequence included in thedeposited clone, which is hybridizable to SEQ ID NO:1 or SEQ ID NO:3,having biological activity.

The invention further relates to a polynucleotide which is a variant ofSEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to a polynucleotide which is an allelicvariant of SEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:2 or SEQ ID NO:4.

The invention further relates to a polynucleotide which represents thecomplimentary sequence (antisense) of SEQ ID NO:1 or SEQ ID NO:4.

The invention further relates to a polynucleotide capable of hybridizingunder stringent conditions to any one of the polynucleotides specifiedherein, wherein said polynucleotide does not hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence ofonly A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:2 or SEQ ID NO:4, wherein the polynucleotide fragmentcomprises a nucleotide sequence encoding a subtilisin protease Kproteinase.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO: 1 or SEQ ID NO:3, wherein the polynucleotide fragmentcomprises a nucleotide sequence encoding the sequence identified as SEQID NO:2 or SEQ ID NO:4 or the polypeptide encoded by the cDNA sequenceincluded in the deposited clone, which is hybridizable to SEQ ID NO:1 orSEQ ID NO:3.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1 or SEQ ID NO:3, wherein the polynucleotide fragmentcomprises the entire nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3or the cDNA sequence included in the deposited clone, which ishybridizable to SEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1 or SEQ ID NO:3, wherein the nucleotide sequence comprisessequential nucleotide deletions from either the C-terminus or theN-terminus.

The invention further relates to an isolated polypeptide comprising anamino acid sequence that comprises a polypeptide fragment of SEQ ID NO:2or SEQ ID NO:4 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide fragment of SEQ ID NO:2or SEQ ID NO:4 or the encoded sequence included in the deposited clone,having biological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:2 orSEQ ID NO:4 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide epitope of SEQ ID NO:2 orSEQ ID NO:4 or the encoded sequence included in the deposited clone.

The invention further relates to a full length protein of SEQ ID NO:2 orSEQ ID NO:4 or the encoded sequence included in the deposited clone.

The invention further relates to a variant of SEQ ID NO:2 or SEQ IDNO:4.

The invention further relates to an allelic variant of SEQ ID NO:2 orSEQ ID NO:4.

The invention further relates to a species homologue of SEQ ID NO:2 orSEQ ID NO:4.

The invention further relates to the isolated polypeptide of SEQ ID NO:2or SEQ ID NO:4, wherein the full length protein comprises sequentialamino acid deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:2 or SEQ ID NO:4.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, comprising administering to amammalian subject a therapeutically effective amount of the polypeptideof SEQ ID NO:2 or SEQ ID NO:4 or the polynucleotide of SEQ ID NO:1 orSEQ ID NO:3.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of: (a) determining the presence or absence of amutation in the polynucleotide of SEQ ID NO:1 or SEQ ID NO:3; and (b)diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or absence of saidmutation.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of: (a) determining the presence or amount ofexpression of the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 in abiological sample; and (b) diagnosing a pathological condition or asusceptibility to a pathological condition based on the presence oramount of expression of the polypeptide.

The invention further relates to a method for identifying a bindingpartner to the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 comprising thesteps of: (a) contacting the polypeptide of SEQ ID NO:2 or SEQ ID NO:4with a binding partner; and (b) determining whether the binding partnereffects an activity of the polypeptide.

The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:1 or SEQ ID NO:3.

The invention further relates to a method of identifying an activity ina biological assay, wherein the method comprises the steps of: (a)expressing SEQ ID NO:1 or SEQ ID NO:3 in a cell; (b) isolating thesupernatant; (c) detecting an activity in a biological assay; and (d)identifying the protein in the supernatant having the activity.

The invention further relates to a process for making polynucleotidesequences encoding gene products having altered activity selected fromthe group consisting of SEQ ID NO:2 or SEQ ID NO:4 activity comprisingthe steps of: (a) shuffling a nucleotide sequence of SEQ ID NO:1 or SEQID NO:3; (b) expressing the resulting shuffled nucleotide sequences; and(c) selecting for altered activity selected from the group consisting ofSEQ ID NO:2 or SEQ ID NO:4 activity as compared to the activity selectedfrom the group consisting of SEQ ID NO:2 or SEQ ID NO:4 activity of thegene product of said unmodified nucleotide sequence.

The invention further relates to a shuffled polynucleotide sequenceproduced by a shuffling process, wherein said shuffled DNA moleculeencodes a gene product having enhanced tolerance to an inhibitor of anyone of the activities selected from the group consisting of SEQ ID NO:2or SEQ ID NO:4 activity.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2 or SEQ ID NO:4, in addition to, its encoding nucleic acid, whereinthe medical condition is a reproductive disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2 or SEQ ID NO:4, in addition to, its encoding nucleic acid, whereinthe medical condition is a disorder related to aberrant PCSK9 signalingand/or activity.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2 or SEQ ID NO:4, in addition to, its encoding nucleic acid, whereinthe medical condition is a cardiovascular disorder,hypercholesterolemia, autosomal dominant hypercholesterolemia; disordersassociated with aberrant LDL receptor function; disorders associatedwith apolipoprotein B; disorders associated with autosomal recessivehypercholesterolemia; disorders associated with elevated cholesterol;disorders associated with elevated LDL; disorders associated withreduced clearance rate of LDL in the liver, disorders associated withelevated LDL apoB production; familial hypercholesterolemia; lipidmetabolism disorders; elevated LDL; cholesterol depositions; tendonxanthomas; atheroma; premature arteriosclerosis, coronary heart disease;famialial defective apolipoprotein B; statin hypersensitivity; disordersassociated with accelerated LDLR degradation, neural differentiationdisorders.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2 or SEQ ID NO:4, in addition to, its encoding nucleic acid, whereinthe medical condition is a metabolic disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2 or SEQ ID NO:4, in addition to, its encoding nucleic acid, whereinthe medical condition is a metabolic disorder selected from the groupconsisting of: dyslipidemia, diabetic dyslipidemia, mixed dyslipidemia,hypercholesteremia, hypertriglyceridemia, type II diabetes mellitus,type I diabetes, insulin resistance, hyperlipidemia, obesity, anorexianervosa.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of PCSK9b and/or PCSK9c, comprisingthe steps of: (a) combining a candidate modulator compound with PCSK9band/or PCSK9c having the sequence set forth in SEQ ID NO:2 or SEQ IDNO:4; and (b) measuring an effect of the candidate modulator compound onthe activity of PCSK9b and/or PCSK9c.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of PCSK9b and/or PCSK9c, comprisingthe steps of: (a) combining a candidate modulator compound with PCSK9band/or PCSK9c having the sequence set forth in SEQ ID NO:2 or SEQ IDNO:4; and (b) measuring an effect of the candidate modulator compound onthe activity of PCSK9b and/or PCSK9c, wherein said method optionallyincludes the addition of a suitable PCSK9 substrate either before,during, or after addition of said candidate modulator compound.

The invention further relates to a method of identifying an antagonistcompound that modulates the biological activity of PCSK9b and/or PCSK9c,comprising the steps of: (a) combining a candidate modulator compoundwith PCSK9b and/or PCSK9c having the sequence set forth in SEQ ID NO:2or SEQ ID NO:4 in the presence of a suitable PCSK9 substrate; and (b)identifying antagonist compounds by measuring an effect of the candidatemodulator compound on the activity of PCSK9b and/or PCSK9c, wherein saididentified antagonist compound decreases proteinase activity of PCSK9band/or PCSK9c.

The invention further relates to a method of identifying an agonistcompound that modulates the biological activity of PCSK9b and/or PCSK9c,comprising the steps of: (a) combining a candidate modulator compoundwith PCSK9b and/or PCSK9c having the sequence set forth in SEQ ID NO:2or SEQ ID NO:4 in the presence of a suitable PCSK9 substrate; and (b)identifying agonist compounds by measuring an effect of the candidatemodulator compound on the activity of PCSK9b and/or PCSK9c, wherein saididentified agonist compound increases proteinase activity of PCSK9band/or PCSK9c.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of PCSK9b and/or PCSK9c, comprisingthe steps of: (a) combining a candidate modulator compound with a hostcell expressing PCSK9b and/or PCSK9c having the sequence as set forth inSEQ ID NO:2 or SEQ ID NO:4; and (b) measuring an effect of the candidatemodulator compound on the activity of the expressed PCSK9b and/orPCSK9c.

The invention further relates to a method of identifying an antagonistcompound that modulates the biological activity of wild-type PCSK9,comprising the steps of: (a) combining a candidate modulator compoundwith PCSK9b and/or PCSK9c having the sequence set forth in SEQ ID NO:2or SEQ ID NO:4 in the presence of a suitable PCSK9 substrate; and (b)identifying antagonist compounds by measuring an effect of the candidatemodulator compound on the activity of PCSK9, wherein said identifiedantagonist compound decreases proteinase activity of PCSK9.

The invention further relates to a method of identifying an agonistcompound that modulates the biological activity of PCSK9, comprising thesteps of: (a) combining a candidate modulator compound with PCSK9band/or PCSK9c having the sequence set forth in SEQ ID NO:2 or SEQ IDNO:4 in the presence of a suitable PCSK9 substrate; and (b) identifyingagonist compounds by measuring an effect of the candidate modulatorcompound on the activity of PCSK9, wherein said identified agonistcompound increases proteinase activity of PCSK9.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of PCSK9, comprising the steps of: (a)combining a candidate modulator compound with a host cell expressingPCSK9b and/or PCSK9c having the sequence as set forth in SEQ ID NO:2 orSEQ ID NO:4; and (b) measuring an effect of the candidate modulatorcompound on the activity of the expressed PCSK9b and/or PCSK9c.

The invention further relates to a method of screening for a compoundthat is capable of modulating the biological activity of PCSK9b and/orPCSK9c, comprising the steps of: (a) providing a host cell describedherein; (b) determining the biological activity of PCSK9b and/or PCSK9cin the absence of a modulator compound; (c) contacting the cell with themodulator compound; and (d) determining the biological activity ofPCSK9b and/or PCSK9c in the presence of the modulator compound; whereina difference between the activity of PCSK9b and/or PCSK9c in thepresence of the modulator compound and in the absence of the modulatorcompound indicates a modulating effect of the compound.

The invention further relates to a method of screening for a compoundthat is capable of modulating the biological activity of PCSK9,comprising the steps of: (a) providing a host cell comprising PCSK9band/or PCSK9c; (b) determining the biological activity of PCSK9 in theabsence of a modulator compound; (c) contacting the cell with themodulator compound; and (d) determining the biological activity of PCSK9in the presence of the modulator compound; wherein a difference betweenthe activity of PCSK9 in the presence of the modulator compound and inthe absence of the modulator compound indicates a modulating effect ofthe compound.

The invention further relates to an N-terminal truncation of PCSK9 (SEQID NO:5), wherein said N-terminal truncation results in the deletion ofanywhere between about 1 to about 218 amino acids from the N-terminus ofSEQ ID NO:5, and wherein said N-terminal truncation results in elevatedPCSK9 biological activity, including, but not limited to decreased LDLRprotein levels, and/or decreased LDL uptake by LDLR.

The invention further relates to an N-terminal truncation of PCSK9 (SEQID NO:5), wherein said N-terminal truncation results in the deletion ofanywhere between about 1 to about 218 amino acids from the N-terminus ofSEQ ID NO:5, including, but not limited to decreased LDLR proteinlevels, and/or decreased LDL uptake by LDLR, and wherein said elevatedPCSK9 biological activity is at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, ormore than wildtype elevated PCSK9 biological activity. In this context,the term “about” shall be construed to mean anywhere between 1, 2, 3, 4,or 5 percent more or less than the cited amount. Alternatively, saidelevated PCSK9 biological activity may be at least about 1×, 2×, 3×, 4×,5×, 6×, 7×, 8×, 9×, or 10× more than wildtype PCSK9 biological activity.In this context, the term “about” shall be construed to mean anywherebetween 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, or 0.9× more orless than the cited amount.

As used herein the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein of PCSK9b and/or PCSK9c.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

The file of this patent contains at least one Figure executed in color.Copies of this patent with color Figure(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1A-IC show the polynucleotide sequence (SEQ ID NO: 1) and deducedamino acid sequence (SEQ ID NO:2) of the novel PCSK9 variant, PCSK9b ofthe present invention. The standard one-letter abbreviation for aminoacids is used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 3175 nucleotides (SEQ IDNO: 1), encoding a polypeptide of 315 amino acids (SEQ ID NO:2). Ananalysis of the PCSK9b polypeptide determined that it comprised thefollowing features: a catalytic domain located from about amino acid 10to about amino acid 256 of SEQ ID NO:2 denoted in italics, with thecanonical catalytic triad residing at amino acids D17, H57, and S217 ofSEQ ID NO:2 denoted by double underlining; and six conserved cysteineresidues located at amino acids C54, C86, C132, C154, C189, and C206 ofSEQ ID NO:2 denoted in bold, with disulfite bonds predicted to formbetween the following cysteine pairs: C54 and C86, and C154 and C189;and a predicted Ca²⁺ ion binding domain predicted to form betweenresidues D191, P162, and V164 of SEQ ID NO:2, denoted by an asterisk (*)below the amino acid residue.

FIG. 2A-2D show the polynucleotide sequence (SEQ ID NO:3) and deducedamino acid sequence (SEQ ID NO:4) of the novel PCSK9 variant, PCSK9c ofthe present invention. The standard one-letter abbreviation for aminoacids is used to illustrate the deduced amino acid sequence. Thepolynucleotide sequence contains a sequence of 3756 nucleotides (SEQ IDNO:3), encoding a polypeptide of 523 amino acids (SEQ ID NO:4). Ananalysis of the PCSK9c polypeptide determined that it comprised thefollowing features: a catalytic domain located from about amino acid 10to about amino acid 256 of SEQ ID NO:4 denoted in italics, with thecanonical catalytic triad residing at amino acids D17, H57, and S217 ofSEQ ID NO:4 denoted by double underlining; and six conserved cysteineresidues located at amino acids C54, C86, C132, C154, C189, and C206 ofSEQ ID NO:4 denoted in bold, with disulfide bonds predicted to formbetween the following cysteine pairs: C54 and C86, and C154 and C189;and a predicted Ca²⁺ ion binding domain predicted to form betweenresidues D191, P162, and V164 of SEQ ID NO:4, denoted by an asterisk (*)below the amino acid residue.

FIG. 3A-3C show the regions of identity and similarity between thesequences of the encoded PCSK9b and PCSK9c polypeptides of the presentinvention with the sequence of the human wild type PCSK9 protein (PCSK9;GENBANK® Accession No: gi|NM_174936; SEQ ID NO:5); as well as thesequence of a known variant of the wild type PCSK9 polypeptide (PCSK9variant; GENBANK® Accession No: gi|AK124635; SEQ ID NO:6). The alignmentwas performed using the CLUSTALW algorithm using default parameters asdescribed herein (VECTOR NTI® suite of programs). The darkly shadedamino acids represent regions of matching identity. The lightly shadedamino acids represent regions of matching similarity. Dots (“”) betweenresidues indicate gapped regions of non-identity for the alignedpolypeptides. The location of the conserved catalytic triad amino acidsare denoted by an asterisk (*).

FIG. 4 shows a table illustrating the percent identity and percentsimilarity between the PCSK9b and PCSK9c polypeptides of the presentinvention with PCSK9 and its variant. As shown, the catalytic domain ofboth PCSK9b and PCSK9c shares 100% identity with the catalytic domain ofPCSK9. The percent identity and percent similarity values weredetermined using the CLUSTALW algorithm using default parameters asdescribed herein (VECTOR NTI® suite of programs).

FIG. 5 provides a schematic diagram of the PCSK9b and PCSK9c variants incomparison to the wild type PCSK9. As shown, both PCSK9b and PCSK9cvariants start from original intron 3. PCSK9b has a novel splicing sitein exon 9 which causes a frame shift and early truncation of theC-terminal domain as a consequence of an in-frame stop codon.

FIG. 6 shows an expression profile of the human PCSK9b and PCSK9cpolypeptides. The figure illustrates the relative expression level ofPCSK9b and PCSK9c amongst various mRNA tissue sources. The identity ofeach tissue is provided in Table IV in Example 3. As shown, the PCSK9band PCSK9c polypeptides were expressed predominately in cerebellum ofthe brain and liver, at levels approximately 3500 fold higher than thelowest expressed tissue. Significant expression was observed in thelung, in pulmonary blood vessels, and tissues of the GI tract.Expression data was obtained by measuring the steady state PCSK9b andPCSK9c mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:10 and 11, and TAQMAN® probe (SEQ ID NO:12) asdescribed in Example 3 herein.

FIG. 7 shows the results of overexpression of PCSK9b and PCSK9c variantsin HEK and CHO cells. Panel “A” shows the observed expression level ofPCSK9, while Panel “B” shows the observed expression level of LDLR,after HEK cells were transiently transfected with wild type PCSK9 (WT),PCSK9 mutant D374Y, and PCSK9b and PCSK9c plasmids. PCSK9b variantplasmids are represented as “#3114”, “#3115”, while the PCSK9c variantplasmids are represented as “#3116” and “#3117”). Expression data wasobtained by measuring the steady state PCSK9b and PCSK9c mRNA levels byquantitative PCR as described in Example 4 herein.

FIG. 8A-8B shows the results of western blot analysis of both celllysates and conditioned media of HEK and CHO cells transientlytransfected with PCSK9b and PCSK9c variants using PCSK9 specificantibody. Panel “A” shows the Western blot of PCSK9 in cell lysates fromPCSK9 transfected HEK cells using PCSK9 antibody, while Panel “B” showsthe Western blot of PCSK9 of conditioned media from CHO cellstransiently transfected with PCSK9 variants. PCSK9b variant plasmids arerepresented as “#3114”, “#3115”, while the PCSK9c variant plasmids arerepresented as “#3116” and “#3117”). Wildtype PCSK9 is represented as“WT”, while the PCSK9 mutant D374Y is represented as “D374Y”. As shown,both PCSK9b and PCSK9c variant proteins were secreted by the transfectedcells as evidenced by their detection in conditioned media despite bothvariants lacking a signal peptide. Western blots were performedaccording to the method described in Example 4 herein.

FIG. 9A-9B shows the results of experiments designed to assess whetherPCSK9b and PCSK9c variants are able to decrease LDL binding to the LDLR.Panel A shows the results of DiI-LDL uptake in HepG2 cells transfectedwith PCSK9b and PCSK9c, while Panel B shows the results of Western bloton cell lysates from CHO cells transfected with PCSK9b or PCSK9c. BothPCSK9 and LDLR antibodies were used for the Western Blots. PCSK9bvariant plasmids are represented as “3114”, “3115”; the PCSK9c variantplasmids are represented as “3116” and “3117”; wildtype PCSK9 isrepresented as “WT”; the PCSK9 mutant D374Y is represented as “D3”;vector only is represented as “vec”, and cells incubated in 10% FBScontaining medium is represented as “FBS”, while cell incubated in 5%lipoprotein deficient serum growth medium is represented as “LPDS”. Asshown in panel “A”, transient transfection of HepG2 cells with bothPCSK9b and PCSK9c variant proteins resulted in a decreased level ofDiI-LDL uptake that exceeded the level observed for wildtype PCSK9. Asshown in panel “B”, transient transfection of CHO cells with the PCSK9cvariant resulted in decreased LDLR protein level, while transienttransfection of variant b did not appear to affect LDLR protein levelunder these conditions. DiI-LDL uptake assays and Western blots wereperformed according to the methods described in Example 4 herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein.

The invention provides novel human sequences that encode variants ofPCSK9 thereof, PCSK9b and PCSK9c, in addition to N-terminal truncatedforms of PCSK9. PCSK9 has been implicated in the incidence of a varietyof diseases and/or disorders, including hypercholesterolemia, itsrelated cardiovascular disorders, in addition to other disorders knownin the art or described herein.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In specific embodiments, the polynucleotides of the invention are aboutat least 15, at least 30, at least 50, at least 100, at least 125, atleast 500, at least 837, at least 903, at least 1000, or at least 1554continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, inlength. In this context, about means 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides longer or shorter at either the 5′ or 3′ end, or both. In afurther embodiment, polynucleotides of the invention comprise a portionof the coding sequences, as disclosed herein, but do not comprise all ora portion of any intron. In another embodiment, the polynucleotidescomprising coding sequences do not contain coding sequences of a genomicflanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). Inother embodiments, the polynucleotides of the invention do not containthe coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15,10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:1 or SEQ ID NO:3, or the cDNAcontained within the clone deposited with the ATCC®. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without a signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ IDNO:1 or SEQ ID NO:3 was generated by overlapping sequences contained inone or more clones (contig analysis). Representative clones containingall of the sequence for SEQ ID NO:1 and SEQ ID NO:3 was deposited withthe American Type Culture Collection (“ATCC®”). As shown in Table I,each clone is identified by a cDNA Clone ID (Identifier) and the ATCC®Deposit Number. The ATCC® is located at 10801 University Boulevard,Manassas, Va. 20110-2209, USA. The ATCC® deposit was made pursuant tothe terms of the Budapest Treaty on the international recognition of thedeposit of microorganisms for purposes of patent procedure. Thedeposited clone for PCSK9b is inserted in the pSport2 vector(Invitrogen). The deposited clone for PCSK9c is inserted in the pSport1vector (Invitrogen).

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373, preferably a Model 3700, from AppliedBiosystems, Inc.), and all amino acid sequences of polypeptides encodedby DNA molecules determined herein were predicted by translation of aDNA sequence determined above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-C (SEQ ID NO: 1), a nucleic acid molecule of the presentinvention encoding the PCSK9b polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material.

Using the information provided herein, such as the nucleotide sequencein FIGS. 2A-D (SEQ ID NO:3), a nucleic acid molecule of the presentinvention encoding the PCSK9c polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:1, the complementthereof, or the cDNA within the clone deposited with the ATCC®.“Stringent hybridization conditions” refers to an overnight incubationat 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ ID NO:Y”refers to a polypeptide sequence, both sequences are identified by aninteger specified in Table I.

“A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

The term “organism” as referred to herein is meant to encompass anyorganism referenced herein, though preferably to eukaryotic organisms,more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that described by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7; 766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are usefulas probes for the identification and isolation of full-length cDNAsand/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present inventionmay comprise one, two, three, four, five, six, seven, eight, or moremembrane domains.

Also, in preferred embodiments the present invention provides methodsfor further refining the biological function of the polynucleotidesand/or polypeptides of the present invention.

Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

In preferred embodiments, the invention provides methods for identifyingthe glycosylation sites inherent in the polynucleotides and polypeptidesof the invention, and the subsequent alteration, deletion, and/oraddition of said sites for a number of desirable characteristics whichinclude, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In further preferred embodiments, methods are provided for evolving thepolynucleotides and polypeptides of the present invention usingmolecular evolution techniques in an effort to create and identify novelvariants with desired structural, functional, and/or physicalcharacteristics.

The present invention further provides for other experimental methodsand procedures currently available to derive functional assignments.These procedures include but are not limited to spotting of clones onarrays, micro-array technology, PCR based methods (e.g., quantitativePCR), anti-sense methodology, gene knockout experiments, and otherprocedures that could use sequence information from clones to build aprimer or a hybrid partner.

Polynucleotides and Polypeptides of the Invention Features of thePolypeptide Encoded by Polynucleotide No:1

The polypeptide of this polynucleotide provided as SEQ ID NO:2 (FIGS.1A-C), encoded by the polynucleotide sequence according to SEQ ID NO:1(FIGS. 1A-C), and/or encoded by the polynucleotide contained within thedeposited clone, PCSK9b (also referred to as PCSK9-b), is a variant ofthe human PCSK9 polypeptide (PCSK9; GENBANK® Accession No: gi|NM_174936;SEQ ID NO:5). An alignment of the PCSK9b polypeptide with PCSK9 inaddition to a known PCSK9 variant (PCSK9 variant; GENBANK® Accession No:gi|AK124635; SEQ ID NO:6) is provided in FIGS. 3A-C. The percentidentity and similarity values between the PCSK9b polypeptide to thesepolypeptides is provided in FIG. 4.

The determined nucleotide sequence of the PCSK9b cDNA in FIGS. 1A-C(SEQID NO:1) contains an open reading frame encoding a protein of about 315amino acid residues, with a deduced molecular weight of about 33.2 kDa.The amino acid sequence of the predicted PCSK9b polypeptide is shown inFIGS. 1A-C(SEQ ID NO:2).

The PCSK9b polypeptide was predicted to comprise a catalytic domainlocated from about amino acid 10 to about amino acid 256 of SEQ ID NO:2,with the canonical catalytic triad residing at amino acids D17, H57, andS217 of SEQ ID NO:2; and six conserved cysteine residues located atamino acids C54, C86, C132, C154, C189, and C206 of SEQ ID NO:2, withdisulfite bonds predicted to form between the following cysteine pairs:C54 and C86, and C154 and C189; and a predicted Ca²⁺ ion binding domainpredicted to form between residues D191, P162, and V164 of SEQ ID NO:2.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-terminal and/or C-terminalboundaries of the above referenced amino acid locations.

Since the PCSK9b polypeptide retains the catalytic triad of thewild-type PCSK9 polypeptide, in addition to its conserved cysteines, itis expected that the PCSK9b polypeptide retains at least some PCSK9biological activity, including but not limited to proteinase activity,convertase activity, subtilisin-kexin isozyme-1/site 1 proteaseactivity, autocatalytic activity cleaving the PCSK9, PCSK9b, PCSK9c, orother variants of PCSK9 between amino acids corresponding to amino acidsGin-151 and Ser-152; sterol-dependent gene expression regulatoryactivity (Maxwell et al., J Lipid Res. 2003; 44: 2109-2119),insulin-dependent gene expression regulatory activity (Shimomura et al.,Proc Natl Acad Sci USA. 1999; 96: 13656-13661), LXR transcriptionfactor-dependent gene expression regulatory activity (Repa et al., GenesDev. 2000; 14: 2819-2830); LDL receptor protein regulatory activity(Maxwell et al., Proc Natl Acad Sci USA. 2004; 101: 7100-7105);statin-dependent upregulation activity (Dubuc et al., ArteriosclerThromb Vasc Biol. 2004; 24: 1454-1459).

In confirmation of PCSK9b retaining PCSK9 biological activity, DiI-LDLuptake assays were performed and PCSK9b was shown to have PCSK9activity. Suprisingly, PCSK9b was found to have greater activity thanwildtype PCSK9. The DiI-LDL uptake assay is a standardized functionalassay for the LDLR receptor, and wildtype PCSK9 activity acts to reduceLDLR activity. Therefore DiI-LDL uptake by cells can be used as asurrogate functional assay for measuring PCSK9 activity. Transientexpression of PCSK9b acted to decrease the uptake of DiI-LDL in HepG2cells, compared to vector control, indicating that PCSK9b is competentto express PCSK9 functional activity on LDLR (FIG. 9). In this assay,PCSK9b showed greater activity than wild-type PCSK9, though not as greatas PCSK9c. Accordingly, PCSK9b retains the ability of wildtype PCSK9 tomodulate the functional activity on LDLR.

PCSK9b was shown to have about 25% more function than wildtype PCSK9 asdemonstrated in the LDL uptake assay (see FIG. 9A). Accordingly, theinvention further relates to an N-terminal truncation of PCSK9, such asPCSK9c for example (SEQ ID NO:2), wherein said N-terminal truncationresults in an elevation of PCSK9 biological activity, including, but notlimited to decreased LDLR protein levels and/or decreased LDL uptake byLDLR, and wherein said elevated PCSK9 biological activity is at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 100%, or more than wildtype elevated PCSK9biological activity. In this context, the term “about” shall beconstrued to mean anywhere between 1, 2, 3, 4, or 5 percent more or lessthan the cited amount. Alternatively, said elevated PCSK9 biologicalactivity may be at least about 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or10× more than wildtype PCSK9 biological activity. In this context, theterm “about” shall be construed to mean anywhere between 0.1×, 0.2×,0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, or 0.9× more or less than the citedamount.

In preferred embodiments, the present invention also encompasses apolynucleotide that comprises a polypeptide that encodes at least about279 contiguous amino acids of SEQ ID NO:2. The present invention alsoencompasses a polynucleotide that comprises at least about 837contiguous nucleotides of SEQ ID NO: 1. In this context, the term“about” shall be construe to mean 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 more or less amino acids at eitherthe N- or C-terminus, or both, or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at either the 5 primeor 3 prime end, or both. Preferably, the polypeptides and/orpolypeptides encoded by said polynucleotides retain biological activity.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, themethinone of the resulting encoded polypeptide of PCSK9b. Specifically,the present invention encompasses the polynucleotide corresponding tonucleotides 253 thru 1194 of SEQ ID NO:1, and the polypeptidecorresponding to amino acids 2 thru 315 of SEQ ID NO:2. Also encompassedare recombinant vectors comprising said encoding sequence, and hostcells comprising said vector.

As described herein, misense mutations of the wild-type PCSK9 at aminoacid locations S127R, F216L, and D374Y have been shown to result inaberrant function of the PCSK9 protein resulting in the incidence ofhypercholesterolemia (Attie, A. D., Art. Thromb. and Vasc. Biol., 2004;24:1337). According, the same mutations at the corresponding amino acidpositions of the PCSK9b polypeptide of the present invention would beuseful in methods of diagnosing patients susceptible to the incidence ofhypercholesterolemia. It should be noted that PCSK9b lacks acorresponding amino acid for the S127R mutation on account ofalternative splicing (see alignment provided in FIGS. 3A-C).

In preferred embodiments, the present invention also encompasses apolynucleotide of SEQ ID NO:2 wherein the serine at amino acid position47 is substituted with a leucine. The present invention also encompassesa polypeptide of SEQ ID NO:2 lacking the initiating start codon, inaddition to, the methionine of the resulting encoded polypeptide ofPCSK9b, wherein serine at amino acid position 47 is substituted with aleucine. Polynucleotides encoding these polypeptides are also provided.Also encompassed are recombinant vectors comprising said encodingsequence, and host cells comprising said vector.

In preferred embodiments, the present invention also encompasses apolynucleotide of SEQ ID NO:2 wherein the aspartic acid at amino acidposition 205 is substituted with a tyrosine. The present invention alsoencompasses a polypeptide of SEQ ID NO:2 lacking the initiating startcodon, in addition to, the methionine of the resulting encodedpolypeptide of PCSK9b, wherein the aspartic acid at amino acid position205 is substituted with a tyrosine. Polynucleotides encoding thesepolypeptides are also provided. Also encompassed are recombinant vectorscomprising said encoding sequence, and host cells comprising saidvector.

Since the PCSK9b polypeptide represents a variant form of the wild-typePCSK9 polypeptide (PCSK9; GENBANK® Accession No: gi|NM_174936; SEQ IDNO:5), it is expected that the expression pattern of the PCSK9b variantis the same or similar to the PCSK9 polypeptide.

The wild-type PCSK9 polypeptide was determined to be predominantlyexpressed in liver and neuronal tissue, and to a lesser extent in kidneymesenchymal cells and intestinal epithelia (Seidah et al., PNAS100(3):928-933 (2003)).

Expression profiling designed to measure the steady state mRNA levelsencoding the PCSK9b and PCSK9c polypeptides confirmed that they sharedthe predominate liver and neuronal expression pattern of wild-typePCSK9. Specifically, PCSK9b and PCSK9c were predominately expressed inliver and cerebellum at levels that were over 3500 times that of thetissue with the lowest expression (the heart). PCSK9b and PCSK9c werealso expressed relatively highly in the lung and its associatedvasculature. PCSK9b and PCSK9c were also expressed throughout thegastrointestinal tract (See FIG. 6). The PCSK9b and PCSK9c expression inthe cerebellum indicates that PCSK9b and PCSK9c may function in neuronaldifferentiation.

The PCSK9b polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing diseases and/or disorders: autosomal dominanthypercholesterolemia; disorders associated with aberrant LDL receptorfunction; disorders associated with apolipoprotein B; disordersassociated with autosomal recessive hypercholesterolemia; disordersassociated with elevated cholesterol; disorders associated with elevatedLDL; disorders associated with reduced clearance rate of LDL in theliver; disorders associated with elevated LDL apoB production; familialhypercholesterolemia; lipid metabolism disorders; elevated LDL;cholesterol depositions; tendon xanthomas; atheroma; prematurearteriosclerosis, coronary heart disease; famialial defectiveapolipoprotein B; statin hypersensitivity; disorders associated withaccelerated LDLR degradation.

The PCSK9b polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing diseases and/or disorders: neural differentiation disorders.

The PCSK9b polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing cardiovascular diseases and/or disorders: myocardioinfarction, congestive heart failure, arrthymias, cardiomyopathy,atherosclerosis, arterialsclerosis, microvascular disease, embolism,thromobosis, pulmonary edema, palpitation, dyspnea, angina, hypotension,syncope, heart murmur, aberrant ECG, hypertrophic cardiomyopathy, theMarfan syndrome, sudden death, prolonged QT syndrome, congenitaldefects, cardiac viral infections, valvular heart disease, hypertension,among others disclosed herein, particularly in the “CardiovascularDisorders” section and below.

Similarly, PCSK9b polynucleotides and polypeptides may be useful forameliorating cardiovascular diseases and symptoms which resultindirectly from various non-cardiovascular effects, which include, butare not limited to, the following, obesity, smoking, Down syndrome(associated with endocardial cushion defect); bony abnormalities of theupper extremities (associated with atrial septal defect in the Holt-Oramsyndrome); muscular dystrophies (associated with cardiomyopathy);hemochromatosis and glycogen storage disease (associated with myocardialinfiltration and restrictive cardiomyopathy); congenital deafness(associated with prolonged QT interval and serious cardiac arrhythmias);Raynaud's disease (associated with primary pulmonary hypertension andcoronary vasospasm); connective tissue disorders, i.e., the Marfansyndrome, Ehlers-Danlos and Hurler syndromes, and related disorders ofmucopolysaccharide metabolism (aortic dilatation, prolapsed mitralvalve, a variety of arterial abnormalities); acromegaly (hypertension,accelerated coronary atherosclerosis, conduction defects,cardiomyopathy); hyperthyroidism (heart failure, atrial fibrillation);hypothyroidism (pericardial effusion, coronary artery disease);rheumatoid arthritis (pericarditis, aortic valve disease); scleroderma(cor pulmonale, myocardial fibrosis, pericarditis); systemic lupuserythematosus (valvulitis, myocarditis, pericarditis); sarcoidosis(arrhythmias, cardiomyopathy); postmenopausal effects, Chlamydialinfections, polycystic ovary disease, thyroid disease, alcoholism, diet,and exfoliative dermatitis (high-output heart failure), for example.

Moreover, polynucleotides and polypeptides, including fragments and/orantagonists thereof, have uses which include, directly or indirectly,treating, preventing, diagnosing, and/or prognosing the following,non-limiting, cardiovascular infections: blood stream invasion,bacteremia, sepsis, Streptococcus pneumoniae infection, group astreptococci infection, group b streptococci infection, Enterococcusinfection, nonenterococcal group D streptococci infection,nonenterococcal group C streptococci infection, nonenterococcal group Gstreptococci infection, Streptoccus viridans infection, Staphylococcusaureus infection, coagulase-negative staphylococci infection,gram-negative Bacilli infection, Enterobacteriaceae infection,Pseudomonas spp. Infection, Acinobacter spp. Infection, Flavobacteriummeningosepticum infection, Aeromonas spp. Infection, Stenotrophomonasmaltophilia infection, gram-negative coccobacilli infection, Haemophilusinfluenza infection, Branhamella catarrhalis infection, anaerobeinfection, Bacteroides fragilis infection, Clostridium infection, fungalinfection, Candida spp. Infection, non-albicans Candida spp. Infection,Hansenula anomala infection, Malassezia furfur infection, nontuberculousMycobacteria infection, Mycobacterium avium infection, Mycobacteriumchelonae infection, Mycobacterium fortuitum infection, spirochetalinfection, Borrelia burgdorferi infection, in addition to any othercardiovascular disease and/or disorder (e.g., non-sepsis) implicated bythe causative agents listed above or elsewhere herein.

The PCSK9b polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing metabolic diseases and/or disorders: dyslipidemia, diabeticdyslipidemia, mixed dyslipidemia, hypercholesteremia,hypertriglyceridemia, type II diabetes mellitus, type I diabetes,insulin resistance, hyperlipidemia, obesity, and/or anorexia nervosa.

The present invention also encompasses therapeutic combinations of amodulator of PCSK9b with a statin for the treatment, prevention and/oramelioration of a disease or disorder referenced herein, particularlydyslipidemia. Representative statins include, but are not limited to,the following: pravastatin, lovastatin, cerivastatin, simvastatin,pitivastatin, atorvastatin or rousuvastatin.

The PCSK9b polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includemodulating signal transduction activity, in various cells, tissues, andorganisms, and particularly in liver, brain, mammalian adipose, omentum,spleen, inflammatory tissues, macrophages, neutrophils, synovialhistiomonocytes, neutrophils, and epithelioid histiocytes.

The PCSK9b polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing hepatic disorders: hepatoblastoma, jaundice, hepatitis, livermetabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, and angiosarcoma,granulomatous liver disease, liver transplantation, hyperbilirubinemia,jaundice, parenchymal liver disease, portal hypertension, hepatobiliarydisease, hepatic parenchyma, hepatic fibrosis, anemia, gallstones,cholestasis, carbon tetrachloride toxicity, beryllium toxicity, vinylchloride toxicity, choledocholithiasis, hepatocellular necrosis,aberrant metabolism of amino acids, aberrant metabolism ofcarbohydrates, aberrant synthesis proteins, aberrant synthesis ofglycoproteins, aberrant degradation of proteins, aberrant degradation ofglycoproteins, aberrant metabolism of drugs, aberrant metabolism ofhormones, aberrant degradation of drugs, aberrant degradation of drugs,aberrant regulation of lipid metabolism, aberrant regulation ofcholesterol metabolism, aberrant glycogenesis, aberrant glycogenolysis,aberrant glycolysis, aberrant gluconeogenesis, hyperglycemia, glucoseintolerance, hyperglycemia, decreased hepatic glucose uptake, decreasedhepatic glycogen synthesis, hepatic resistance to insulin,portal-systemic glucose shunting, peripheral insulin resistance,hormonal abnormalities, increased levels of systemic glucagon, decreasedlevels of systemic cortisol, increased levels of systemic insulin,hypoglycemia, decreased gluconeogenesis, decreased hepatic glycogencontent, hepatic resistance to glucagon, elevated levels of systemicaromatic amino acids, decreased levels of systemic branched-chain aminoacids, hepatic encephalopathy, aberrant hepatic amino acidtransamination, aberrant hepatic amino acid oxidative deamination,aberrant ammonia synthesis, aberant albumin secretion, hypoalbuminemia,aberrant cytochromes b5 function, aberrant P450 function, aberrantglutathione S-acyltransferase function, aberrant cholesterol synthesis,and aberrant bile acid synthesis.

Moreover, polynucleotides and polypeptides, including fragments and/orantagonists thereof, have uses which include, directly or indirectly,treating, preventing, diagnosing, and/or prognosing the following,non-limiting, hepatic infections: liver disease caused by sepsisinfection, liver disease caused by bacteremia, liver disease caused byPneomococcal pneumonia infection, liver disease caused by Toxic shocksyndrome, liver disease caused by Listeriosis, liver disease caused byLegionnaries' disease, liver disease caused by Brucellosis infection,liver disease caused by Neisseria gonorrhoeae infection, liver diseasecaused by Yersinia infection, liver disease caused by Salmonellosis,liver disease caused by Nocardiosis, liver disease caused by Spirocheteinfection, liver disease caused by Treponema pallidum infection, liverdisease caused by Brrelia burgdorferi infection, liver disease caused byLeptospirosis, liver disease caused by Coxiella burnetii infection,liver disease caused by Rickettsia richettsii infection, liver diseasecaused by Chlamydia trachomatis infection, liver disease caused byChlamydia psittaci infection, liver disease caused by hepatitis virusinfection, liver disease caused by Epstein-Barr virus infection inaddition to any other hepatic disease and/or disorder implicated by thecausative agents listed above or elsewhere herein.

The PCSK9b polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include identification ofmodulators of PCSK9b function including antibodies (for detection orneutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains of the PCSK9b protein could be used asdiagnostic agents of lipid metabolic disorders, includinghypercholesterolemia, among others.

The expression level of PCSK9b also may be useful as a biomarker forpredicting which patients may be at risk of developinghypercholesterolemia, and/or those patients which may be at risk ofbeing overly sensitive to statin therapy.

PCSK9b polypeptides and polynucleotides have additional uses whichinclude diagnosing diseases related to the over and/or under expressionof PCSK9b by identifying mutations in the PCSK9b gene by using PCSK9bsequences as probes or by determining PCSK9b protein or mRNA expressionlevels. PCSK9b polypeptides may be useful for screening compounds thataffect the activity of the protein. PCSK9b peptides can also be used forthe generation of specific antibodies and as bait in yeast two hybridscreens to find proteins the specifically interact with PCSK9b(described elsewhere herein).

In preferred embodiments, the present invention is also directed topolynucleotides comprising, or alternatively consisting of, a sequenceencoding the following N-terminal PCSK9b deletion polypeptides: M1-R315,S2-R315, P3-R315, W4-R315, K5-R315, D6-R315, G7-R315, G8-R315, S9-R315,L10-R315, V11-R315, E12-R315, V13-R315, Y14-R315, L15-R315, L16-R315,D17-R315, T18-R315, S19-R315, I20-R315, Q21-R315, S22-R315, D23-R315,H24-R315, R25-R315, E26-R315, I27-R315, E28-R315, G29-R315, R30-R315,V31-R315, M32-R315, V33-R315, T34-R315, D35-R315, F36-R315, E37-R315,N38-R315, V39-R315, P40-R315, E41-R315, E42-R315, D43-R315, G44-R315,T45-R315, R46-R315, F47-R315, H48-R315, R49-R315, Q50-R315, A51-R315,S52-R315, K53-R315, C54-R315, D55-R315, S56-R315, H57-R315, G58-R315,T59-R315, H60-R315, L61-R315, A62-R315, G63-R315, V64-R315, V65-R315,S66-R315, G67-R315, R68-R315, D69-R315, A70-R315, G71-R315, V72-R315,A73-R315, K74-R315, G75-R315, A76-R315, S77-R315, M78-R315, R79-R315,S80-R315, L81-R315, R82-R315, V83-R315, L84-R315, N85-R315, C86-R315,Q87-R315, G88-R315, K89-R315, G90-R315, T91-R315, V92-R315, S93-R315,G94-R315, T95-R315, L96-R315, I97-R315, G98-R315, L99-R315, E100-R315,F101-R315, I102-R315, R103-R315, K104-R315, S105-R315, Q106-R315,L107-R315, V108-R315, Q109-R315, P110-R315, V111-R315, G112-R315,P113-R315, L114-R315, V115-R315, V116-R315, L117-R315, L118-R315,P119-R315, L120-R315, A121-R315, G122-R315, G123-R315, Y124-R315,S125-R315, R126-R315, V127-R315, L128-R315, N129-R315, A130-R315,A131-R315, C132-R315, Q133-R315, R134-R315, L135-R315, A136-R315,R137-R315, A138-R315, G139-R315, V140-R315, V141-R315, L142-R315,V143-R315, T144-R315, A145-R315, A146-R315, G147-R315, N148-R315,F149-R315, R150-R315, D151-R315, D152-R315, A153-R315, C154-R315,L155-R315, Y156-R315, S157-R315, P158-R315, A159-R315, S160-R315,A161-R315, P162-R315, E163-R315, V164-R315, I165-R315, T166-R315,V167-R315, G168-R315, A169-R315, T170-R315, N171-R315, A172-R315,Q173-R315, D174-R315, Q175-R315, P176-R315, V177-R315, T178-R315,L179-R315, G180-R315, T181-R315, L182-R315, G183-R315, T184-R315,N185-R315, F186-R315, G187-R315, R188-R315, C189-R315, V190-R315,D191-R315, L192-R315, F193-R315, A194-R315, P195-R315, G196-R315,E197-R315, D198-R315, I199-R315, I200-R315, G201-R315, A202-R315,S203-R315, S204-R315, D205-R315, C206-R315, S207-R315, T208-R315,C209-R315, F210-R315, V211-R315, S212-R315, Q213-R315, S214-R315,G215-R315, T216-R315, S217-R315, Q218-R315, A219-R315, A220-R315,A221-R315, H222-R315, V223-R315, A224-R315, G225-R315, I226-R315,A227-R315, A228-R315, M229-R315, M230-R315, L231-R315, S232-R315,A233-R315, E234-R315, P235-R315, E236-R315, L237-R315, T238-R315,L239-R315, A240-R315, E241-R315, L242-R315, R243-R315, Q244-R315,R245-R315, L246-R315, I247-R315, H248-R315, F249-R315, S250-R315,A251-R315, K252-R315, D253-R315, V254-R315, I255-R315, N256-R315,E257-R315, A258-R315, W259-R315, F260-R315, P261-R315, E262-R315,D263-R315, Q264-R315, R265-R315, V266-R315, L267-R315, T268-R315,P269-R315, N270-R315, L271-R315, V272-R315, A273-R315, A274-R315,L275-R315, P276-R315, P277-R315, S278-R315, T279-R315, H280-R315,G281-R315, A282-R315, G283-R315, P284-R315, F285-R315, C286-R315,R287-R315, L288-R315, A289-R315, A290-R315, V291-R315, L292-R315,Q293-R315, D294-R315, C295-R315, V296-R315, V297-R315, S298-R315,T299-R315, L300-R315, G301-R315, A302-R315, Y303-R315, T304-R315,D305-R315, G306-R315, H307-R315, S308-R315, and/or H309-R315 of SEQ IDNO:2. Polypeptide sequences encoded by these polynucleotides are alsoprovided. In addition, the invention also encompasses polynucleotidesencoding a polypeptide that is at least as long as any one of theaforementioned polypeptides. The present invention also encompasses theuse of these N-terminal PCSK9b deletion polypeptides as immunogenicand/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the present invention is also directed topolynucleotides comprising, or alternatively consisting of, a sequenceencoding the following C-terminal PCSK9b deletion polypeptides: M1-R315,M1-P314, M1-R313, M1-L312, M1-P311, M1-R310, M1-H309, M1-S308, M1-H307,M1-G306, M1-D305, M1-T304, M1-Y303, M1-A302, M1-G301, M1-L300, M1-T299,M1-S298, M1-V297, M1-V296, M1-C295, M1-D294, M1-Q293, M1-L292, M1-V291,M1-A290, M1-A289, M1-L288, M1-R287, M1-C286, M1-F285, M1-P284, M1-G283,M1-A282, M1-G281, M1-H280, M1-T279, M1-S278, M1-P277, M1-P276, M1-L275,M1-A274, M1-A273, M1-V272, M1-L271, M1-N270, M1-P269, M1-T268, M1-L267,M1-V266, M1-R265, M1-Q264, M1-D263, M1-E262, M1-P261, M1-F260, M1-W259,M1-A258, M1-E257, M1-N256, M1-1255, M1-V254, M1-D253, M1-K252, M1-A251,M1-S250, M1-F249, M1-H248, M1-1247, M1-L246, M1-R245, M1-Q244, M1-R243,M1-L242, M1-E241, M1-A240, M1-L239, M1-T238, M1-L237, M1-E236, M1-P235,M1-E234, M1-A233, M1-S232, M1-L231, M1-M230, M1-M229, M1-A228, M1-A227,M1-I226, M1-G225, M1-A224, M1-V223, M1-H222, M1-A221, M1-A220, M1-A219,M1-Q218, M1-S217, M1-T216, M1-G215, M1-S214, M1-Q213, M1-S212, M1-V211,M1-F210, M1-C209, M1-T208, M1-S207, M1-C206, M1-D205, M1-S204, M1-S203,M1-A202, M1-G201, M1-I200, M1-I199, M1-D198, M1-E197, M1-G196, M1-P195,M1-A194, M1-F193, M1-L192, M1-D191, M1-V190, M1-C189, M1-R188, M1-G187,M1-F186, M1-N185, M1-T184, M1-G183, M1-L182, M1-T181, M1-G180, M1-L179,M1-T178, M1-V177, M1-P176, M1-Q175, M1-D174, M1-Q173, M1-A172, M1-N171,M1-T170, M1-A169, M1-G168, M1-V167, M1-T166, M1-I165, M1-V164, M1-E163,M1-P162, M1-A161, M1-S160, M1-A159, M1-P158, M1-S157, M1-Y156, M1-L55,M1-C154, M1-A153, M1-D152, M1-D151, M1-R150, M1-F149, M1-N148, M1-G147,M1-A146, M1-A145, M1-T144, M1-V143, M1-L142, M1-V141, M1-V140, M1-G139,M1-A138, M1-R137, M1-A136, M1-L135, M1-R134, M1-Q133, M1-C132, M1-A131,M1-A130, M1-N129, M1-L128, M1-V127, M1-R126, M1-S125, M1-Y124, M1-G123,M1-G122, M1-A121, M1-L120, M1-P119, M1-L118, M1-L117, M1-V116, M1-V115,M1-L14, M1-P113, M1-G112, M1-V111, M1-P110, M1-Q109, M1-V108, M1-L107,M1-Q106, M1-S105, M1-K104, M1-R103, M1-I102, M1-F101, M1-E100, M1-L99,M1-G98, M1-197, M1-L96, M1-T95, M1-G94, M1-S93, M1-V92, M1-T91, M1-G90,M1-K89, M1-G88, M1-Q87, M1-C86, M1-N85, M1-L84, M1-V83, M1-R82, M1-L81,M1-S80, M1-R79, M1-M78, M1-S77, M1-A76, M1-G75, M1-K74, M1-A73, M1-V72,M1-G71, M1-A70, M1-D69, M1-R68, M1-G67, M1-S66, M1-V65, M1-V64, M1-G63,M1-A62, M1-L61, M1-H60, M1-T59, M1-G58, M1-H57, M1-S56, M1-D55, M1-C54,M1-K53, M1-S52, M1-A51, M1-Q50, M1-R49, M1-H48, M1-F47, M1-R46, M1-T45,M1-G44, M1-D43, M1-E42, M1-E41, M1-P40, M1-V39, M1-N38, M1-E37, M1-F36,M1-D35, M1-T34, M1-V33, M1-M32, M1-V31, M1-R30, M1-G29, M1-E28, M1-I27,M1-E26, M1-R25, M1-H24, M1-D23, M1-S22, M1-Q21, M1-I20, M1-S19, M1-T18,M1-D17, M1-L16, M1-L15, M1-Y14, M1-V13, M1-E12, M1-V11, M1-L10, M1-S9,M1-G8, and/or M1-G7 of SEQ ID NO:2. Polypeptide sequences encoded bythese polynucleotides are also provided. In addition, the invention alsoencompasses polynucleotides encoding a polypeptide that is at least aslong as any one of the aforementioned polypeptides. The presentinvention also encompasses the use of these C-terminal PCSK9b deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

Alternatively, preferred polypeptides of the present invention maycomprise polypeptide sequences corresponding to, for example, internalregions of the PCSK9b polypeptide (e.g., any combination of both N- andC-terminal PCSK9b polypeptide deletions) of SEQ ID NO:2. For example,internal regions could be defined by the equation: amino acid NX toamino acid CX, wherein NX refers to any N-terminal deletion polypeptideamino acid of PCSK9b (SEQ ID NO:2), and where CX refers to anyC-terminal deletion polypeptide amino acid of PCSK9b (SEQ ID NO:2).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the PCSK9b polypeptide.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 3161 ofSEQ ID NO:1, b is an integer between 15 to 3175, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:2

The polypeptide of this polynucleotide provided as SEQ ID NO:4 (FIGS.2A-D), encoded by the polynucleotide sequence according to SEQ ID NO:3(FIGS. 2A-D), and/or encoded by the polynucleotide contained within thedeposited clone, PCSK9c (also referred to as PCSK9-c), is a variant ofthe human PCSK9 polypeptide (PCSK9; GENBANK® Accession No: gi|NM_174936;SEQ ID NO:5). An alignment of the PCSK9c polypeptide with PCSK9 inaddition to a known PCSK9 variant (PCSK9 variant; GENBANK® Accession No:gi|AK124635; SEQ ID NO:6) is provided in FIGS. 3A-C. The percentidentity and similarity values between the PCSK9c polypeptide to thesepolypeptides is provided in FIG. 4.

The determined nucleotide sequence of the PCSK9c cDNA in FIGS. 2A-D (SEQID NO:3) contains an open reading frame encoding a protein of about 523amino acid residues, with a deduced molecular weight of about 55.2 kDa.The amino acid sequence of the predicted PCSK9c polypeptide is shown inFIGS. 2A-D (SEQ ID NO:4).

The PCSK9c polypeptide was predicted to comprise a catalytic domainlocated from about amino acid 10 to about amino acid 256 of SEQ ID NO:4,with the canonical catalytic triad residing at amino acids D17, H57, andS217 of SEQ ID NO:4; and six conserved cysteine residues located atamino acids C54, C86, C132, C154, C189, and C206 of SEQ ID NO:4, withdisulfide bonds predicted to form between the following cysteine pairs:C54 and C86, and C154 and C189; and a predicted Ca²⁺ ion binding domainpredicted to form between residues D191, P162, and V164 of SEQ ID NO:4.In this context, the term “about” may be construed to mean 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids beyond the N-terminal and/or C-terminalboundaries of the above referenced amino acid locations.

Since the PCSK9c polypeptide retains the catalytic triad of thewild-type PCSK9 polypeptide, in addition to its conserved cysteines, itis expected that the PCSK9c polypeptide retains PCSK9 biologicalactivity, including but not limited to proteinase activity, convertaseactivity, subtilisin-kexin isozyme-1/site 1 protease activity,autocatalytic activity cleaving the PCSK9, PCSK9b, PCSK9c, or othervariants of PCSK9 between amino acids corresponding to amino acidsGin-151 and Ser-152; sterol-dependent gene expression regulatoryactivity (Maxwell et al., J Lipid Res. 2003; 44: 2109-2119),insulin-dependent gene expression regulatory activity (Shimomura et al.,Proc Natl Acad Sci USA. 1999; 96: 13656-13661), LXR transcriptionfactor-dependent gene expression regulatory activity (Repa et al., GenesDev. 2000; 14: 2819-2830); LDL receptor protein regulatory activity(Maxwell et al., Proc Natl Acad Sci USA. 2004; 101: 7100-7105);statin-dependent upregulation activity (Dubuc et al., ArieriosclerThromb Vasc Biol. 2004; 24: 1454-1459).

In confirmation of PCSK9c retaining PCSK9 biological activity, DiI-LDLuptake assays were performed and PCSK9c was shown to have PCSK9activity. Suprisingly, PCSK9c was found to have greater activity thanwildtype PCSK9. The DiI-LDL uptake assay is a standardized functionalassay for the LDLR receptor, and wildtype PCSK9 activity acts to reduceLDLR activity. Therefore DiI-LDL uptake by cells can be used as asurrogate functional assay for measuring PCSK9 activity. Transientexpression of PCSK9c acted to decrease the uptake of DiI-LDL in HepG2cells, compared to vector control, indicating that PCSK9c is competentto express PCSK9 functional activity on LDLR (FIG. 9). In this assay,PCSK9c showed greater activity than both wild-type PCSK9 and PCSK9b.Accordingly, PCSK9c retains the ability of wildtype PCSK9 to modulatethe functional activity on LDLR.

PCSK9c was shown to have about 50% more function than wildtype PCSK9 asdemonstrated in the LDL uptake assay (see FIG. 9A). Accordingly, theinvention further relates to an N-terminal truncation of PCSK9, such asPCSK9c for example (SEQ ID NO:4), wherein said N-terminal truncationresults in an elevation of PCSK9 biological activity, including, but notlimited to decreased LDLR protein levels and/or decreased LDL uptake byLDLR, and wherein said elevated PCSK9 biological activity is at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 100%, or more than wildtype elevated PCSK9biological activity. In this context, the term “about” shall beconstrued to mean anywhere between 1, 2, 3, 4, or 5 percent more or lessthan the cited amount. Alternatively, said elevated PCSK9 biologicalactivity may be at least about 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or10× more than wildtype PCSK9 biological activity. In this context, theterm “about” shall be construed to mean anywhere between 0.1×, 0.2×,0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, or 0.9× more or less than the citedamount.

In preferred embodiments, the present invention also encompasses apolynucleotide that comprises a polypeptide that encodes at least about301 contiguous amino acids of SEQ ID NO:4. The present invention alsoencompasses a polypeptide that comprises at least about 496 amino acidsof SEQ ID NO:4. The present invention also encompasses a polypeptidethat comprises at least about 518 amino acids of SEQ ID NO:4. Thepresent invention also encompasses a polynucleotide that comprises atleast about 903 contiguous nucleotides of SEQ ID NO:3. The presentinvention also encompasses a polynucleotide that comprises at leastabout 1488 contiguous nucleotides of SEQ ID NO:3. The present inventionalso encompasses a polynucleotide that comprises at least about 1554contiguous nucleotides of SEQ ID NO:3. In this context, the term “about”shall be construe to mean 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 more or less amino acids at either the N-or C-terminus, or both, or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nucleotides at either the 5 prime or 3prime end, or both. Preferably, the polypeptides and/or polypeptidesencoded by said polynucleotides retain biological activity.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to themethionine of the resulting encoded polypeptide of PCSK9c. Specifically,the present invention encompasses the polynucleotide corresponding tonucleotides 884 thru 2049 of SEQ ID NO:3, and the polypeptidecorresponding to amino acids 2 thru 523 of SEQ ID NO:4. Also encompassedare recombinant vectors comprising said encoding sequence, and hostcells comprising said vector.

Since the PCSK9c polypeptide represents a variant form of the wild-typePCSK9 polypeptide (PCSK9; GENBANK® Accession No: gi|NM_174936; SEQ IDNO:5), it is expected that the expression pattern of the PCSK9c variantis the same or similar to the PCSK9 polypeptide.

As described herein, misense mutations of the wild-type PCSK9 at aminoacid locations S127R, F216L, and D374Y have been shown to result inaberrant function of the PCSK9 protein resulting in the incidence ofhypercholesterolemia (Attie, A. D., Art. Thromb. and Vasc. Biol., 2004;24:1337). According, the same mutations at the corresponding amino acidpositions of the PCSK9c polypeptide of the present invention would beuseful in methods of diagnosing patients susceptible to the incidence ofhypercholesterolemia. It should be noted that PCSK9c lacks acorresponding amino acid for the S127R mutation on account ofalternative splicing (see alignment provided in FIGS. 3A-C).

In preferred embodiments, the present invention also encompasses apolynucleotide of SEQ ID NO:4 wherein the serine at amino acid position47 is substituted with a leucine. The present invention also encompassesa polypeptide of SEQ ID NO:4 lacking the initiating start codon, inaddition to, the methionine of the resulting encoded polypeptide ofPCSK9c, wherein serine at amino acid position 47 is substituted with aleucine. Polynucleotides encoding these polypeptides are also provided.Also encompassed are recombinant vectors comprising said encodingsequence, and host cells comprising said vector.

In preferred embodiments, the present invention also encompasses apolynucleotide of SEQ ID NO:4 wherein the aspartic acid at amino acidposition 205 is substituted with a tyrosine. The present invention alsoencompasses a polypeptide of SEQ ID NO:4 lacking the initiating startcodon, in addition to, the methionine of the resulting encodedpolypeptide of PCSK9c, wherein aspartic acid at amino acid position 205is substituted with a tyrosine. Polynucleotides encoding thesepolypeptides are also provided. Also encompassed are recombinant vectorscomprising said encoding sequence, and host cells comprising saidvector.

The wild-type PCSK9 polypeptide was determined to be predominantlyexpressed in liver and neuronal tissue, and to a lesser extent in kidneymesenchymal cells and intestinal epithelia (Seidah et al., PNAS100(3):928-933 (2003)).

Expression profiling designed to measure the steady state mRNA levelsencoding the PCSK9b and PCSK9c polypeptides confirmed that they sharedthe predominate liver and neuronal expression pattern of wild-typePCSK9. Specifically, PCSK9b and PCSK9c were predominately expressed inliver and cerebellum at levels that were over 3500 times that of thetissue with the lowest expression (the heart). PCSK9b and PCSK9c werealso expressed relatively highly in the lung and its associatedvasculature. PCSK9b and PCSK9c were also expressed throughout thegastrointestinal tract (See FIG. 6). The PCSK9b and PCSK9c expression inthe cerebellum indicates that PCSK9b and PCSK9c may function in neuronaldifferentiation.

The PCSK9c polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing diseases and/or disorders: autosomal dominanthypercholesterolemia; disorders associated with aberrant LDL receptorfunction; disorders associated with apolipoprotein B; disordersassociated with autosomal recessive hypercholesterolemia; disordersassociated with elevated cholesterol; disorders associated with elevatedLDL; disorders associated with reduced clearance rate of LDL in theliver, disorders associated with elevated LDL apoB production; familialhypercholesterolemia; lipid metabolism disorders; elevated LDL;cholesterol depositions; tendon xanthomas; atheroma; prematurearteriosclerosis, coronary heart disease; famialial defectiveapolipoprotein B; statin hypersensitivity; disorders associated withaccelerated LDLR degradation.

The PCSK9c polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing diseases and/or disorders: neural differentiation disorders.

The PCSK9c polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing cardiovascular diseases and/or disorders: myocardioinfarction, congestive heart failure, arrthymias, cardiomyopathy,atherosclerosis, arterialsclerosis, microvascular disease, embolism,thromobosis, pulmonary edema, palpitation, dyspnea, angina, hypotension,syncope, heart murmur, aberrant ECG, hypertrophic cardiomyopathy, theMarfan syndrome, sudden death, prolonged QT syndrome, congenitaldefects, cardiac viral infections, valvular heart disease, hypertension,among others disclosed herein, particularly in the “CardiovascularDisorders” section and below.

Similarly, PCSK9c polynucleotides and polypeptides may be useful forameliorating cardiovascular diseases and symptoms which resultindirectly from various non-cardiovascular effects, which include, butare not limited to, the following, obesity, smoking, Down syndrome(associated with endocardial cushion defect); bony abnormalities of theupper extremities (associated with atrial septal defect in the Holt-Oramsyndrome); muscular dystrophies (associated with cardiomyopathy);hemochromatosis and glycogen storage disease (associated with myocardialinfiltration and restrictive cardiomyopathy); congenital deafness(associated with prolonged QT interval and serious cardiac arrhythmias);Raynaud's disease (associated with primary pulmonary hypertension andcoronary vasospasm); connective tissue disorders, i.e., the Marfansyndrome, Ehlers-Danlos and Hurler syndromes, and related disorders ofmucopolysaccharide metabolism (aortic dilatation, prolapsed mitralvalve, a variety of arterial abnormalities); acromegaly (hypertension,accelerated coronary atherosclerosis, conduction defects,cardiomyopathy); hyperthyroidism (heart failure, atrial fibrillation);hypothyroidism (pericardial effusion, coronary artery disease);rheumatoid arthritis (pericarditis, aortic valve disease); scleroderma(cor pulmonale, myocardial fibrosis, pericarditis); systemic lupuserythematosus (valvulitis, myocarditis, pericarditis); sarcoidosis(arrhythmias, cardiomyopathy); postmenopausal effects, Chlamydialinfections, polycystic ovary disease, thyroid disease, alcoholism, diet,and exfoliative dermatitis (high-output heart failure), for example.

Moreover, polynucleotides and polypeptides, including fragments and/orantagonists thereof, have uses which include, directly or indirectly,treating, preventing, diagnosing, and/or prognosing the following,non-limiting, cardiovascular infections: blood stream invasion,bacteremia, sepsis, Streptococcus pneumoniae infection, group astreptococci infection, group b streptococci infection, Enterococcusinfection, nonenterococcal group D streptococci infection,nonenterococcal group C streptococci infection, nonenterococcal group Gstreptococci infection, Streptoccus viridans infection, Staphylococcusaureus infection, coagulase-negative staphylococci infection,gram-negative Bacilli infection, Enterobacteriaceae infection,Pseudomonas spp. Infection, Acinobacter spp. Infection, Flavobacteriummeningosepticum infection, Aeromonas spp. Infection, Stenotrophomonasmaltophilia infection, gram-negative coccobacilli infection, Haemophilusinfluenza infection, Branhamella catarrhalis infection, anaerobeinfection, Bacteroides fragilis infection, Clostridium infection, fungalinfection, Candida spp. Infection, non-albicans Candida spp. Infection,Hansenula anomala infection, Malassezia furfur infection, nontuberculousMycobacteria infection, Mycobacterium avium infection, Mycobacteriumchelonae infection, Mycobacterium fortuitum infection, spirochetalinfection, Borrelia burgdorferi infection, in addition to any othercardiovascular disease and/or disorder (e.g., non-sepsis) implicated bythe causative agents listed above or elsewhere herein.

The PCSK9c polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing metabolic diseases and/or disorders: dyslipidemia, diabeticdyslipidemia, mixed dyslipidemia, hypercholesteremia,hypertriglyceridemia, type II diabetes mellitus, type I diabetes,insulin resistance, hyperlipidemia, obesity, and/or anorexia nervosa.

The present invention also encompasses therapeutic combinations of amodulator of PCSK9c with a statin for the treatment, prevention and/oramelioration of a disease or disorder referenced herein, particularlydyslipidemia. Representative statins include, but are not limited to,the following: pravastatin, lovastatin, cerivastatin, simvastatin,pitivastatin, atorvastatin or rousuvastatin.

The PCSK9c polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includemodulating signal transduction activity, in various cells, tissues, andorganisms, and particularly in liver, brain, mammalian adipose, omentum,spleen, inflammatory tissues, macrophages, neutrophils, synovialhistiomonocytes, neutrophils, and epithelioid histiocytes.

The PCSK9c polynucleotides and polypeptides of the present invention,including modulators and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing hepatic disorders: hepatoblastoma, jaundice, hepatitis, livermetabolic diseases and conditions that are attributable to thedifferentiation of hepatocyte progenitor cells, cirrhosis, hepaticcysts, pyrogenic abscess, amebic abcess, hydatid cyst,cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma,hepatocellulae carcinoma, cholangiocarcinoma, and angiosarcoma,granulomatous liver disease, liver transplantation, hyperbilirubinemia,jaundice, parenchymal liver disease, portal hypertension, hepatobiliarydisease, hepatic parenchyma, hepatic fibrosis, anemia, gallstones,cholestasis, carbon tetrachloride toxicity, beryllium toxicity, vinylchloride toxicity, choledocholithiasis, hepatocellular necrosis,aberrant metabolism of amino acids, aberrant metabolism ofcarbohydrates, aberrant synthesis proteins, aberrant synthesis ofglycoproteins, aberrant degradation of proteins, aberrant degradation ofglycoproteins, aberrant metabolism of drugs, aberrant metabolism ofhormones, aberrant degradation of drugs, aberrant degradation of drugs,aberrant regulation of lipid metabolism, aberrant regulation ofcholesterol metabolism, aberrant glycogenesis, aberrant glycogenolysis,aberrant glycolysis, aberrant gluconeogenesis, hyperglycemia, glucoseintolerance, hyperglycemia, decreased hepatic glucose uptake, decreasedhepatic glycogen synthesis, hepatic resistance to insulin,portal-systemic glucose shunting, peripheral insulin resistance,hormonal abnormalities, increased levels of systemic glucagon, decreasedlevels of systemic cortisol, increased levels of systemic insulin,hypoglycemia, decreased gluconeogenesis, decreased hepatic glycogencontent, hepatic resistance to glucagon, elevated levels of systemicaromatic amino acids, decreased levels of systemic branched-chain aminoacids, hepatic encephalopathy, aberrant hepatic amino acidtransamination, aberrant hepatic amino acid oxidative deamination,aberrant ammonia synthesis, aberant albumin secretion, hypoalbuminemia,aberrant cytochromes b5 function, aberrant P450 function, aberrantglutathione S-acyltransferase function, aberrant cholesterol synthesis,and aberrant bile acid synthesis.

Moreover, polynucleotides and polypeptides, including fragments and/orantagonists thereof, have uses which include, directly or indirectly,treating, preventing, diagnosing, and/or prognosing the following,non-limiting, hepatic infections: liver disease caused by sepsisinfection, liver disease caused by bacteremia, liver disease caused byPneomococcal pneumonia infection, liver disease caused by Toxic shocksyndrome, liver disease caused by Listeriosis, liver disease caused byLegionnaries' disease, liver disease caused by Brucellosis infection,liver disease caused by Neisseria gonorrhoeae infection, liver diseasecaused by Yersinia infection, liver disease caused by Salmonellosis,liver disease caused by Nocardiosis, liver disease caused by Spirocheteinfection, liver disease caused by Treponema pallidum infection, liverdisease caused by Brrelia burgdorferi infection, liver disease caused byLeptospirosis, liver disease caused by Coxiella burnetii infection,liver disease caused by Rickettsia richettsii infection, liver diseasecaused by Chlamydia trachomatis infection, liver disease caused byChlamydia psittaci infection, liver disease caused by hepatitis virusinfection, liver disease caused by Epstein-Barr virus infection inaddition to any other hepatic disease and/or disorder implicated by thecausative agents listed above or elsewhere herein.

The PCSK9c polynucleotides and polypeptides, including fragments and/orantagonsists thereof, may have uses which include identification ofmodulators of PCSK9c function including antibodies (for detection orneutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains of the PCSK9c protein could be used asdiagnostic agents of lipid metabolic disorders, includinghypercholesterolemia, among others.

The expression level of PCSK9c also may be useful as a biomarker forpredicting which patients may be at risk of developinghypercholesterolemia, and/or those patients which may be at risk ofbeing overly sensitive to statin therapy.

PCSK9c polypeptides and polynucleotides have additional uses whichinclude diagnosing diseases related to the over and/or under expressionof PCSK9c by identifying mutations in the PCSK9c gene by using PCSK9csequences as probes or by determining PCSK9c protein or mRNA expressionlevels. PCSK9c polypeptides may be useful for screening compounds thataffect the activity of the protein. PCSK9c peptides can also be used forthe generation of specific antibodies and as bait in yeast two hybridscreens to find proteins the specifically interact with PCSK9c(described elsewhere herein).

In preferred embodiments, the present invention is also directed topolynucleotides comprising, or alternatively consisting of, a sequenceencoding following N-terminal PCSK9c deletion polypeptides: M1-Q523,S2-Q523, P3-Q523, W4-Q523, K5-Q523, D6-Q523, G7-Q523, G8-Q523, S9-Q523,L10-Q523, V11-Q523, E12-Q523, V13-Q523, Y14-Q523, L15-Q523, L16-Q523,D17-Q523, T18-Q523, S19-Q523, I20-Q523, Q21-Q523, S22-Q523, D23-Q523,H24-Q523, R25-Q523, E26-Q523, I127-Q523, E28-Q523, G29-Q523, R30-Q523,V31-Q523, M32-Q523, V33-Q523, T34-Q523, D35-Q523, F36-Q523, E37-Q523,N38-Q523, V39-Q523, P40-Q523, E41-Q523, E42-Q523, D43-Q523, G44-Q523,T45-Q523, R46-Q523, F47-Q523, H48-Q523, R49-Q523, Q50-Q523, A51-Q523,S52-Q523, K53-Q523, C54-Q523, D55-Q523, S56-Q523, H57-Q523, G58-Q523,T59-Q523, H60-Q523, L61-Q523, A62-Q523, G63-Q523, V64-Q523, V65-Q523,S66-Q523, G67-Q523, R68-Q523, D69-Q523, A70-Q523, G71-Q523, V72-Q523,A73-Q523, K74-Q523, G75-Q523, A76-Q523, S77-Q523, M78-Q523, R79-Q523,S80-Q523, L81-Q523, R82-Q523, V83-Q523, L84-Q523, N85-Q523, C86-Q523,Q87-Q523, G88-Q523, K89-Q523, G90-Q523, T91-Q523, V92-Q523, S93-Q523,G94-Q523, T95-Q523, L96-Q523, I97-Q523, G98-Q523, L99-Q523, E100-Q523,F101-Q523, I102-Q523, R103-Q523, K104-Q523, S105-Q523, Q106-Q523,L107-Q523, V108-Q523, Q109-Q523, P110-Q523, V111-Q523, G112-Q523,P113-Q523, L114-Q523, V115-Q523, V116-Q523, L117-Q523, L118-Q523,P119-Q523, L120-Q523, A121-Q523, G122-Q523, G123-Q523, Y124-Q523,S125-Q523, R126-Q523, V127-Q523, L128-Q523, N129-Q523, A130-Q523,A131-Q523, C132-Q523, Q133-Q523, R134-Q523, L135-Q523, A136-Q523,R137-Q523, A138-Q523, G139-Q523, V140-Q523, V141-Q523, L142-Q523,V143-Q523, T144-Q523, A145-Q523, A146-Q523, G147-Q523, N148-Q523,F149-Q523, R150-Q523, D151-Q523, D152-Q523, A153-Q523, C154-Q523,L155-Q523, Y156-Q523, S157-Q523, P158-Q523, A159-Q523, S160-Q523,A161-Q523, P162-Q523, E163-Q523, V164-Q523, I165-Q523, T166-Q523,V167-Q523, G168-Q523, A169-Q523, T170-Q523, N171-Q523, A172-Q523,Q173-Q523, D174-Q523, Q175-Q523, P176-Q523, V177-Q523, T178-Q523,L179-Q523, G180-Q523, T181-Q523, L182-Q523, G183-Q523, T184-Q523,N185-Q523, F186-Q523, G187-Q523, R188-Q523, C189-Q523, V190-Q523,D191-Q523, L192-Q523, F193-Q523, A194-Q523, P195-Q523, G196-Q523,E197-Q523, D198-Q523, I199-Q523, I200-Q523, G201-Q523, A202-Q523,S203-Q523, S204-Q523, D205-Q523, C206-Q523, S207-Q523, T208-Q523,C209-Q523, F210-Q523, V211-Q523, S212-Q523, Q213-Q523, S214-Q523,G215-Q523, T216-Q523, S217-Q523, Q218-Q523, A219-Q523, A220-Q523,A221-Q523, H222-Q523, V223-Q523, A224-Q523, G225-Q523, I226-Q523,A227-Q523, A228-Q523, M229-Q523, M230-Q523, L231-Q523, S232-Q523,A233-Q523, E234-Q523, P235-Q523, E236-Q523, L237-Q523, T238-Q523,L239-Q523, A240-Q523, E241-Q523, L242-Q523, R243-Q523, Q244-Q523,R245-Q523, L246-Q523, I247-Q523, H248-Q523, F249-Q523, S250-Q523,A251-Q523, K252-Q523, D253-Q523, V254-Q523, I255-Q523, N256-Q523,E257-Q523, A258-Q523, W259-Q523, F260-Q523, P261-Q523, E262-Q523,D263-Q523, Q264-Q523, R265-Q523, V266-Q523, L267-Q523, T268-Q523,P269-Q523, N270-Q523, L271-Q523, V272-Q523, A273-Q523, A274-Q523,L275-Q523, P276-Q523, P277-Q523, S278-Q523, T279-Q523, H280-Q523,G281-Q523, A282-Q523, G283-Q523, W284-Q523, Q285-Q523, L286-Q523,F287-Q523, C288-Q523, R289-Q523, T290-Q523, V291-Q523, W292-Q523,S293-Q523, A294-Q523, H295-Q523, S296-Q523, G297-Q523, P298-Q523,T299-Q523, R300-Q523, M301-Q523, A302-Q523, T303-Q523, A304-Q523,I305-Q523, A306-Q523, R307-Q523, C308-Q523, A309-Q523, P310-Q523,D311-Q523, E312-Q523, E313-Q523, L314-Q523, L315-Q523, S316-Q523,C317-Q523, S318-Q523, S319-Q523, F320-Q523, S321-Q523, R322-Q523,S323-Q523, G324-Q523, K325-Q523, R326-Q523, R327-Q523, G328-Q523,E329-Q523, R330-Q523, M331-Q523, E332-Q523, A333-Q523, Q334-Q523,G335-Q523, G336-Q523, K337-Q523, L338-Q523, V339-Q523, C340-Q523,R341-Q523, A342-Q523, H343-Q523, N344-Q523, A345-Q523, F346-Q523,G347-Q523, G348-Q523, E349-Q523, G350-Q523, V351-Q523, Y352-Q523,A353-Q523, I354-Q523, A355-Q523, R356-Q523, C357-Q523, C358-Q523,L359-Q523, L360-Q523, P361-Q523, Q362-Q523, A363-Q523, N364-Q523,C365-Q523, S366-Q523, V367-Q523, H368-Q523, T369-Q523, A370-Q523,P371-Q523, P372-Q523, A373-Q523, E374-Q523, A375-Q523, S376-Q523,M377-Q523, G378-Q523, T379-Q523, R380-Q523, V381-Q523, H382-Q523,C383-Q523, H384-Q523, Q385-Q523, Q386-Q523, G387-Q523, H388-Q523,V389-Q523, L390-Q523, T391-Q523, G392-Q523, C393-Q523, S394-Q523,S395-Q523, H396-Q523, W397-Q523, E398-Q523, V399-Q523, E400-Q523,D401-Q523, L402-Q523, G403-Q523, T404-Q523, H405-Q523, K406-Q523,P407-Q523, P408-Q523, V409-Q523, L410-Q523, R411-Q523, P412-Q523,R413-Q523, G414-Q523, Q415-Q523, P416-Q523, N417-Q523, Q418-Q523,C419-Q523, V420-Q523, G421-Q523, H422-Q523, R423-Q523, E424-Q523,A425-Q523, S426-Q523, I427-Q523, H428-Q523, A429-Q523, S430-Q523,C431-Q523, C432-Q523, H433-Q523, A434-Q523, P435-Q523, G436-Q523,L437-Q523, E438-Q523, C439-Q523, K440-Q523, V441-Q523, K442-Q523,E443-Q523, H444-Q523, G445-Q523, I446-Q523, P447-Q523, A448-Q523,P449-Q523, Q450-Q523, E451-Q523, Q452-Q523, V453-Q523, T454-Q523,V455-Q523, A456-Q523, C457-Q523, E458-Q523, E459-Q523, G460-Q523,W461-Q523, T462-Q523, L463-Q523, T464-Q523, G465-Q523, C466-Q523,S467-Q523, A468-Q523, L469-Q523, P470-Q523, G471-Q523, T472-Q523,S473-Q523, H474-Q523, V475-Q523, L476-Q523, G477-Q523, A478-Q523,Y479-Q523, A480-Q523, V481-Q523, D482-Q523, N483-Q523, T484-Q523,C485-Q523, V486-Q523, V487-Q523, R488-Q523, S489-Q523, R490-Q523,D491-Q523, V492-Q523, S493-Q523, T494-Q523, T495-Q523, G496-Q523,S497-Q523, T498-Q523, S499-Q523, E500-Q523, G501-Q523, A502-Q523,V503-Q523, T504-Q523, A505-Q523, V506-Q523, A507-Q523, I508-Q523,C509-Q523, C510-Q523, R511-Q523, S512-Q523, R513-Q523, H514-Q523,L515-Q523, A516-Q523, and/or Q517-Q523 of SEQ ID NO:4. Polypeptidesequences encoded by these polynucleotides are also provided. Inaddition, the invention also encompasses polynucleotides encoding apolypeptide that is at least as long as any one of the aforementionedpolypeptides. The present invention also encompasses the use of theseN-terminal PCSK9c deletion polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

In preferred embodiments, the present invention is also directed topolynucleotides comprising, or alternatively consisting of, a sequenceencoding the following C-terminal PCSK9c deletion polypeptides: M1-Q523,M1-L522, M1-E521, M1-Q520, M1-S519, M1-A518, M1-Q517, M1-A516, M1-L515,M1-H514, M1-R513, M1-S512, M1-R511, M1-C510, M1-C509, M1-I508, M1-A507,M1-V506, M1-A505, M1-T504, M1-V503, M1-A502, M1-G501, M1-E500, M1-S499,M1-T498, M1-S497, M1-G496, M1-T495, M1-T494, M1-S493, M1-V492, M1-D491,M1-R490, M1-S489, M1-R488, M1-V487, M1-V486, M1-C485, M1-T484, M1-N483,M1-D482, M1-V481, M1-A480, M1-Y479, M1-A478, M1-G477, M1-L476, M1-V475,M1-H474, M1-S473, M1-T472, M1-G471, M1-P470, M1-L469, M1-A468, M1-S467,M1-C466, M1-G465, M1-T464, M1-L463, M1-T462, M1-W461, M1-G460, M1-E459,M1-E458, M1-C457, M1-A456, M1-V455, M1-T454, M1-V453, M1-Q452, M1-E451,M1-Q450, M1-P449, M1-A448, M1-P447, M1-1446, M1-G445, M1-H444, M1-E443,M1-K442, M1-V441, M1-K440, M1-C439, M1-E438, M1-L437, M1-G436, M1-P435,M1-A434, M1-H433, M1-C432, M1-C431, M1-S430, M1-A429, M1-H428, M1-I427,M1-S426, M1-A425, M1-E424, M1-R423, M1-H422, M1-G421, M1-V420, M1-C419,M1-Q418, M1-N417, M1-P416, M1-Q415, M1-G414, M1-R413, M1-P412, M1-R411,M1-L410, M1-V409, M1-P408, M1-P407, M1-K406, M1-H405, M1-T404, M1-G403,M1-L402, M1-D401, M1-E400, M1-V399, M1-E398, M1-W397, M1-H396, M1-S395,M1-S394, M1-C393, M1-G392, M1-T391, M1-L390, M1-V389, M1-H388, M1-G387,M1-Q386, M1-Q385, M1-H384, M1-C383, M1-H382, M1-V381, M1-R380, M1-T379,M1-G378, M1-M377, M1-S376, M1-A375, M1-E374, M1-A373, M1-P372, M1-P371,M1-A370, M1-T369, M1-H368, M1-V367, M1-S366, M1-C365, M1-N364, M1-A363,M1-Q362, M1-P361, M1-L360, M1-L359, M1-C358, M1-C357, M1-R356, M1-A355,M1-I354, M1-A353, M1-Y352, M1-V351, M1-G350, M1-E349, M1-G348, M1-G347,M1-F346, M1-A345, M1-N344, M1-H343, M1-A342, M1-R341, M1-C340, M1-V339,M1-L338, M1-K337, M1-G336, M1-G335, M1-Q334, M1-A333, M1-E332, M1-M331,M1-R330, M1-E329, M1-G328, M1-R327, M1-R326, M1-K325, M1-G324, M1-S323,M1-R322, M1-S321, M1-F320, M1-S319, M1-S318, M1-C317, M1-S316, M1-L315,M1-L314, M1-E313, M1-E312, M1-D311, M1-P310, M1-A309, M1-C308, M1-R307,M1-A306, M1-I305, M1-A304, M1-T303, M1-A302, M1-M301, M1-R300, M1-T299,M1-P298, M1-G297, M1-S296, M1-H295, M1-A294, M1-S293, M1-W292, M1-V291,M1-T290, M1-R289, M1-C288, M1-F287, M1-L286, M1-Q285, M1-W284, M1-G283,M1-A282, M1-G281, M1-H280, M1-T279, M1-S278, M1-P277, M1-P276, M1-L275,M1-A274, M1-A273, M1-V272, M1-L271, M1-N270, M1-P269, M1-T268, M1-L267,M1-V266, M1-R265, M1-Q264, M1-D263, M1-E262, M1-P261, M1-F260, M1-W259,M1-A258, M1-E257, M1-N256, M1-I255, M1-V254, M1-D253, M1-K252, M1-A251,M1-S250, M1-F249, M1-H248, M1-I247, M1-L246, M1-R245, M1-Q244, M1-R243,M1-L242, M1-E241, M1-A240, M1-L239, M1-T238, M1-L237, M1-E236, M1-P235,M1-E234, M1-A233, M1-S232, M1-L231, M1-M230, M1-M229, M1-A228, M1-A227,M1-I226, M1-G225, M1-A224, M1-V223, M1-H222, M1-A221, M1-A220, M1-A219,M1-Q218, M1-S217, M1-T216, M1-G215, M1-S214, M1-Q213, M1-S212, M1-V211,M1-F210, M1-C209, M1-T208, M1-S207, M1-C206, M1-D205, M1-S204, M1-S203,M1-A202, M1-G201, M1-I200, M1-I199, M1-D198, M1-E197, M1-G196, M1-P195,M1-A194, M1-F193, M1-L192, M1-D191, M1-V190, M1-C189, M1-R188, M1-G187,M1-F186, M1-N185, M1-T184, M1-G183, M1-L182, M1-T181, M1-G180, M1-L179,M1-T178, M1-V177, M1-P176, M1-Q175, M1-D174, M1-Q173, M1-A172, M1-N171,M1-T170, M1-A169, M1-G168, M1-V167, M1-T166, M1-I165, M1-V164, M1-E163,M1-P162, M1-A161, M1-S160, M1-A159, M1-P158, M1-S157, M1-Y156, M1-L55,M1-C154, M1-A153, M1-D152, M1-D151, M1-R50, M1-F149, M1-N148, M1-G147,M1-A146, M1-A145, M1-T144, M1-V143, M1-L142, M1-V141, M1-V140, M1-G139,M1-A138, M1-R137, M1-A136, M1-L135, M1-R134, M1-Q133, M1-C132, M1-A131,M1-A130, M1-N129, M1-L128, M1-V127, M1-R126, M1-S125, M1-Y124, M1-G123,M1-G122, M1-A121, M1-L120, M1-P119, M1-L118, M1-L117, M1-V116, M1-V115,M1-L114, M1-P113, M1-G112, M1-V111, M1-P110, M1-Q109, M1-V108, M1-L107,M1-Q106, M1-S105, M1-K104, M1-R103, M1-I102, M1-F101, M1-E100, M1-L99,M1-G98, M1-I97, M1-L96, M1-T95, M1-G94, M1-S93, M1-V92, M1-T91, M1-G90,M1-K89, M1-G88, M1-Q87, M1-C86, M1-N85, M1-L84, M1-V83, M1-R82, M1-L81,M1-S80, M1-R79, M1-M78, M1-S77, M1-A76, M1-G75, M1-K74, M1-A73, M1-V72,M1-G71, M1-A70, M1-D69, M1-R68, M1-G67, M1-S66, M1-V65, M1-V64, M1-G63,M1-A62, M1-L61, M1-H60, M1-T59, M1-G58, M1-H57, M1-S56, M1-D55, M1-C54,M1-K53, M1-S52, M1-A51, M1-Q50, M1-R49, M1-H48, M1-F47, M1-R46, M1-T45,M1-G44, M1-D43, M1-E42, M1-E41, M1-P40, M1-V39, M1-N38, M1-E37, M1-F36,M1-D35, M1-T34, M1-V33, M1-M32, M1-V31, M1-R30, M1-G29, M1-E28, M1-I27,M1-E26, M1-R25, M1-H24, M1-D23, M1-S22, M1-Q21, M1-I20, M1-S19, M1-T18,M1-D17, M1-L16, M1-L15, M1-Y14, M1-V13, M1-E12, M1-V11, M1-L10, M1-S9,M1-G8, and/or M1-G7 of SEQ ID NO:4. Polypeptide sequences encoded bythese polynucleotides are also provided. In addition, the invention alsoencompasses polynucleotides encoding a polypeptide that is at least aslong as any one of the aforementioned polypeptides. The presentinvention also encompasses the use of these C-terminal PCSK9c deletionpolypeptides as immunogenic and/or antigenic epitopes as describedelsewhere herein.

Alternatively, preferred polypeptides of the present invention maycomprise polypeptide sequences corresponding to, for example, internalregions of the PCSK9c polypeptide (e.g., any combination of both N- andC-terminal PCSK9c polypeptide deletions) of SEQ ID NO:4. For example,internal regions could be defined by the equation: amino acid NX toamino acid CX, wherein NX refers to any N-terminal deletion polypeptideamino acid of PCSK9c (SEQ ID NO:4), and where CX refers to anyC-terminal deletion polypeptide amino acid of PCSK9c (SEQ ID NO:4).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these polypeptides as animmunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the PCSK9c polypeptide.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:3 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 3742 ofSEQ ID NO:3, b is an integer between 15 to 3756, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:3,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:3

The invention further relates to an N-terminal truncation of PCSK9 (SEQID NO:5), wherein said N-terminal truncation results in the deletion ofanywhere between about 1 to about 218 amino acids from the N-terminus ofSEQ ID NO:5, and wherein said N-terminal truncation results in elevatedPCSK9 biological activity, including, but not limited to decreased LDLRprotein levels, and/or decreased LDL uptake by LDLR. Polynucleotidesencoding such PCSK9 truncations are provided as SEQ ID NO:38.

The inventors are the first to discover that N-terminal truncations ofPCSK9 result in elevated levels of PCSK9 biological activity. Suchtruncated forms may also greatly facilitate the identification of smallmolecule modulators of PCSK9 as well. Experiments designed to assesswhether truncation of the N-terminus of PCSK9 results in elevatedbiological activity have been performed. For example, truncation of theN-terminus by 15 amino acids resulted in significant increases in PCSK9activity (data not shown), although not as significant as that observedfor the PCSK9b and PCSK9c variants (see FIGS. 9A-B). Furthermore,truncation was tolerated by PCSK9 up to a truncation of about 218 aminoacids, after which decreased levels of PCSK9 biological activity wasobserved (data not shown).

The invention further relates to an N-terminal truncation of PCSK9 (SEQID NO:5), wherein said N-terminal truncation results in the deletion ofanywhere between about 1 to about 218 amino acids from the N-terminus ofSEQ ID NO:5, including, but not limited to decreased LDLR protein levelsand/or decreased LDL uptake by LDLR, and wherein said elevated PCSK9biological activity is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, or morethan wildtype elevated PCSK9 biological activity. In this context, theterm “about” shall be construed to mean anywhere between 1, 2, 3, 4, or5 percent more or less than the cited amount. Alternatively, saidelevated PCSK9 biological activity may be at least about 1×, 2×, 3×, 4×,5×, 6×, 7×, 8×, 9×, or 10× more than wildtype PCSK9 biological activity.In this context, the term “about” shall be construed to mean anywherebetween 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, or 0.9× more orless than the cited amount.

Preferably, an N-terminal deletion mutant of PCSK9 (SEQ ID NO:5) is atleast about 15 amino acids, but less than about 218 amino acids, of SEQID NO:5. Truncated forms of PCSK9 may be created using molecular biologytechniques (see Example 13), proteolytic cleavage, post-translationalprocessing, chemical synthesis, etc., among other methods known in theart.

The coding region of PCSK9 encoding polynucleotides is represented bynucleotides 292 to 2367 of SEQ ID NO:38.

In preferred embodiments, the present invention is also directed topolynucleotides comprising, or alternatively consisting of, a sequenceencoding the following N-terminal PCSK9c deletion polypeptides: M1-Q692,G2-Q692, T3-Q692, V4-Q692, S5-Q692, S6-Q692, R7-Q692, R8-Q692, S9-Q692,W10-Q692, W11-Q692, P12-Q692, L13-Q692, P14-Q692, L15-Q692, L16-Q692,L17-Q692, L18-Q692, L19-Q692, L20-Q692, L21-Q692, L22-Q692, L23-Q692,G24-Q692, P25-Q692, A26-Q692, G27-Q692, A28-Q692, R29-Q692, A30-Q692,Q31-Q692, E32-Q692, D33-Q692, E34-Q692, D35-Q692, G36-Q692, D37-Q692,Y38-Q692, E39-Q692, E40-Q692, L41-Q692, V42-Q692, L43-Q692, A44-Q692,L45-Q692, R46-Q692, S47-Q692, E48-Q692, E49-Q692, D50-Q692, G51-Q692,L52-Q692, A53-Q692, E54-Q692, A55-Q692, P56-Q692, E57-Q692, H58-Q692,G59-Q692, T60-Q692, T61-Q692, A62-Q692, T63-Q692, F64-Q692, H65-Q692,R66-Q692, C67-Q692, A68-Q692, K69-Q692, D70-Q692, P71-Q692, W72-Q692,R73-Q692, L74-Q692, P75-Q692, G76-Q692, T77-Q692, Y78-Q692, V79-Q692,V80-Q692, V81-Q692, L82-Q692, K83-Q692, E84-Q692, E85-Q692, T86-Q692,H87-Q692, L88-Q692, S89-Q692, Q90-Q692, S91-Q692, E92-Q692, R93-Q692,T94-Q692, A95-Q692, R96-Q692, R97-Q692, L98-Q692, Q99-Q692, A100-Q692,Q101-Q692, A102-Q692, A103-Q692, R104-Q692, R105-Q692, G106-Q692,Y107-Q692, L108-Q692, T109-Q692, K110-Q692, I111-Q692, L112-Q692,H113-Q692, V114-Q692, F115-Q692, H116-Q692, G117-Q692, L18-Q692,L119-Q692, P120-Q692, G121-Q692, F122-Q692, L123-Q692, V124-Q692,K125-Q692, M126-Q692, S127-Q692, G128-Q692, D129-Q692, L130-Q692,L131-Q692, E132-Q692, L133-Q692, A134-Q692, L135-Q692, K136-Q692,L137-Q692, P138-Q692, H139-Q692, V140-Q692, D141-Q692, Y142-Q692,I143-Q692, E144-Q692, E145-Q692, D146-Q692, S147-Q692, S148-Q692,V149-Q692, F150-Q692, A151-Q692, Q152-Q692, S153-Q692, I154-Q692,P155-Q692, W156-Q692, N157-Q692, L158-Q692, E159-Q692, R160-Q692,I161-Q692, T162-Q692, P163-Q692, P164-Q692, R165-Q692, Y166-Q692,R167-Q692, A168-Q692, D169-Q692, E170-Q692, Y171-Q692, Q172-Q692,P173-Q692, P174-Q692, D175-Q692, G176-Q692, G177-Q692, S178-Q692,L179-Q692, V180-Q692, E181-Q692, V182-Q692, Y183-Q692, L184-Q692,L185-Q692, D186-Q692, T187-Q692, S188-Q692, I189-Q692, Q190-Q692,S191-Q692, D192-Q692, H193-Q692, R194-Q692, E195-Q692, I196-Q692,E197-Q692, G198-Q692, R199-Q692, V200-Q692, M201-Q692, V202-Q692,T203-Q692, D204-Q692, F205-Q692, E206-Q692, N207-Q692, V208-Q692,P209-Q692, E210-Q692, E211-Q692, D212-Q692, G213-Q692, T214-Q692,R215-Q692, F216-Q692, H217-Q692, R218-Q692, Q219-Q692, A220-Q692,S221-Q692, K222-Q692, C223-Q692, D224-Q692, S225-Q692, H226-Q692,G227-Q692, T228-Q692, H229-Q692, L230-Q692, A231-Q692, G232-Q692,V233-Q692, V234-Q692, S235-Q692, G236-Q692, R237-Q692, D238-Q692,A239-Q692, G240-Q692, V241-Q692, A242-Q692, K243-Q692, G244-Q692,A245-Q692, S246-Q692, M247-Q692, R248-Q692, S249-Q692, L250-Q692,R251-Q692, V252-Q692, L253-Q692, N254-Q692, C255-Q692, Q256-Q692,G257-Q692, K258-Q692, G259-Q692, T260-Q692, V261-Q692, S262-Q692,G263-Q692, T264-Q692, L265-Q692, I266-Q692, G267-Q692, L268-Q692,E269-Q692, F270-Q692, I271-Q692, R272-Q692, K273-Q692, S274-Q692,Q275-Q692, L276-Q692, V277-Q692, Q278-Q692, P279-Q692, V280-Q692,G281-Q692, P282-Q692, L283-Q692, V284-Q692, V285-Q692, L286-Q692,L287-Q692, P288-Q692, L289-Q692, A290-Q692, G291-Q692, G292-Q692,Y293-Q692, S294-Q692, R295-Q692, V296-Q692, L297-Q692, N298-Q692,A299-Q692, A300-Q692, C301-Q692, Q302-Q692, R303-Q692, L304-Q692,A305-Q692, R306-Q692, A307-Q692, G308-Q692, V309-Q692, V310-Q692,L311-Q692, V312-Q692, T313-Q692, A314-Q692, A315-Q692, G316-Q692,N317-Q692, F318-Q692, R319-Q692, D320-Q692, D321-Q692, A322-Q692,C323-Q692, L324-Q692, Y325-Q692, S326-Q692, P327-Q692, A328-Q692,S329-Q692, A330-Q692, P331-Q692, E332-Q692, V333-Q692, I334-Q692,T335-Q692, V336-Q692, G337-Q692, A338-Q692, T339-Q692, N340-Q692,A341-Q692, Q342-Q692, D343-Q692, Q344-Q692, P345-Q692, V346-Q692,T347-Q692, L348-Q692, G349-Q692, T350-Q692, L351-Q692, G352-Q692,T353-Q692, N354-Q692, F355-Q692, G356-Q692, R357-Q692, C358-Q692,V359-Q692, D360-Q692, L361-Q692, F362-Q692, A363-Q692, P364-Q692,G365-Q692, E366-Q692, D367-Q692, I368-Q692, I369-Q692, G370-Q692,A371-Q692, S372-Q692, S373-Q692, D374-Q692, C375-Q692, S376-Q692,T377-Q692, C378-Q692, F379-Q692, V380-Q692, S381-Q692, Q382-Q692,S383-Q692, G384-Q692, T385-Q692, S386-Q692, Q387-Q692, A388-Q692,A389-Q692, A390-Q692, H391-Q692, V392-Q692, A393-Q692, G394-Q692,I395-Q692, A396-Q692, A397-Q692, M398-Q692, M399-Q692, L400-Q692,S401-Q692, A402-Q692, E403-Q692, P404-Q692, E405-Q692, L406-Q692,T407-Q692, L408-Q692, A409-Q692, E410-Q692, L411-Q692, R412-Q692,Q413-Q692, R414-Q692, L415-Q692, I416-Q692, H417-Q692, F418-Q692,S419-Q692, A420-Q692, K421-Q692, D422-Q692, V423-Q692, I424-Q692,N425-Q692, E426-Q692, A427-Q692, W428-Q692, F429-Q692, P430-Q692,E431-Q692, D432-Q692, Q433-Q692, R434-Q692, V435-Q692, L436-Q692,T437-Q692, P438-Q692, N439-Q692, L440-Q692, V441-Q692, A442-Q692,A443-Q692, L444-Q692, P445-Q692, P446-Q692, S447-Q692, T448-Q692,H449-Q692, G450-Q692, A451-Q692, G452-Q692, W453-Q692, Q454-Q692,L455-Q692, F456-Q692, C457-Q692, R458-Q692, T459-Q692, V460-Q692,W461-Q692, S462-Q692, A463-Q692, H464-Q692, S465-Q692, G466-Q692,P467-Q692, T468-Q692, R469-Q692, M470-Q692, A471-Q692, T472-Q692,A473-Q692, V474-Q692, A475-Q692, R476-Q692, C477-Q692, A478-Q692,P479-Q692, D480-Q692, E481-Q692, E482-Q692, L483-Q692, L484-Q692,S485-Q692, C486-Q692, S487-Q692, S488-Q692, F489-Q692, S490-Q692,R491-Q692, S492-Q692, G493-Q692, K494-Q692, R495-Q692, R496-Q692,G497-Q692, E498-Q692, R499-Q692, M500-Q692, E501-Q692, A502-Q692,Q503-Q692, G504-Q692, G505-Q692, K506-Q692, L507-Q692, V508-Q692,C509-Q692, R510-Q692, A511-Q692, H512-Q692, N513-Q692, A514-Q692,F515-Q692, G516-Q692, G517-Q692, E518-Q692, G519-Q692, V520-Q692,Y521-Q692, A522-Q692, I523-Q692, A524-Q692, R525-Q692, C526-Q692,C527-Q692, L528-Q692, L529-Q692, P530-Q692, Q531-Q692, A532-Q692,N533-Q692, C534-Q692, S535-Q692, V536-Q692, H537-Q692, T538-Q692,A539-Q692, P540-Q692, P541-Q692, A542-Q692, E543-Q692, A544-Q692,S545-Q692, M546-Q692, G547-Q692, T548-Q692, R549-Q692, V550-Q692,H551-Q692, C552-Q692, H553-Q692, Q554-Q692, Q555-Q692, G556-Q692,H557-Q692, V558-Q692, L559-Q692, T560-Q692, G561-Q692, C562-Q692,S563-Q692, S564-Q692, H565-Q692, W566-Q692, E567-Q692, V568-Q692,E569-Q692, D570-Q692, L571-Q692, G572-Q692, T573-Q692, H574-Q692,K575-Q692, P576-Q692, P577-Q692, V578-Q692, L579-Q692, R580-Q692,P581-Q692, R582-Q692, G583-Q692, Q584-Q692, P585-Q692, N586-Q692,Q587-Q692, C588-Q692, V589-Q692, G590-Q692, H591-Q692, R592-Q692,E593-Q692, A594-Q692, S595-Q692, I596-Q692, H597-Q692, A598-Q692,S599-Q692, C600-Q692, C601-Q692, H602-Q692, A603-Q692, P604-Q692,G605-Q692, L606-Q692, E607-Q692, C608-Q692, K609-Q692, V610-Q692,K611-Q692, E612-Q692, H613-Q692, G614-Q692, I615-Q692, P616-Q692,A617-Q692, P618-Q692, Q619-Q692, E620-Q692, Q621-Q692, V622-Q692,T623-Q692, V624-Q692, A625-Q692, C626-Q692, E627-Q692, E628-Q692,G629-Q692, W630-Q692, T631-Q692, L632-Q692, T633-Q692, G634-Q692,C635-Q692, S636-Q692, A637-Q692, L638-Q692, P639-Q692, G640-Q692,T641-Q692, S642-Q692, H643-Q692, V644-Q692, L645-Q692, G646-Q692,A647-Q692, Y648-Q692, A649-Q692, V650-Q692, D651-Q692, N652-Q692,T653-Q692, C654-Q692, V655-Q692, V656-Q692, R657-Q692, S658-Q692,R659-Q692, D660-Q692, V661-Q692, S662-Q692, T663-Q692, T664-Q692,G665-Q692, S666-Q692, T667-Q692, S668-Q692, E669-Q692, G670-Q692,A671-Q692, V672-Q692, T673-Q692, A674-Q692, V675-Q692, A676-Q692,I677-Q692, C678-Q692, C679-Q692, R680-Q692, S681-Q692, R682-Q692,H683-Q692, L684-Q692, A685-Q692, and/or Q686-Q692 of SEQ ID NO:5.Polypeptide sequences encoded by these polynucleotides are also providedas SEQ ID NO:38. In addition, the invention also encompassespolynucleotides encoding a polypeptide that is at least as long as anyone of the aforementioned polypeptides. The present invention alsoencompasses the use of these N-terminal PCSK9c deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the present invention is also directed topolynucleotides comprising, or alternatively consisting of, a sequenceencoding the following C-terminal PCSK9c deletion polypeptides: M1-Q692,M1-L691, M1-E690, M1-Q689, M1-S688, M1-A687, M1-Q686, M1-A685, M1-L684,M1-H683, M1-R682, M1-S681, M1-R680, M1-C679, M1-C678, M1-1677, M1-A676,M1-V675, M1-A674, M1-T673, M1-V672, M1-A671, M1-G670, M1-E669, M1-S668,M1-T667, M1-S666, M1-G665, M1-T664, M1-T663, M1-S662, M1-V661, M1-D660,M1-R659, M1-S658, M1-R657, M1-V656, M1-V655, M1-C654, M1-T653, M1-N652,M1-D651, M1-V650, M1-A649, M1-Y648, M1-A647, M1-G646, M1-L645, M1-V644,M1-H643, M1-S642, M1-T641, M1-G640, M1-P639, M1-L638, M1-A637, M1-S636,M1-C635, M1-G634, M1-T633, M1-L632, M1-T631, M1-W630, M1-G629, M1-E628,M1-E627, M1-C626, M1-A625, M1-V624, M1-T623, M1-V622, M1-Q621, M1-E620,M1-Q619, M1-P618, M1-A617, M1-P616, M1-1615, M1-G614, M1-H613, M1-E612,M1-K611, M1-V610, M1-K609, M1-C608, M1-E607, M1-L606, M1-G605, M1-P604,M1-A603, M1-H602, M1-C601, M1-C600, M1-S599, M1-A598, M1-H597, M1-1596,M1-S595, M1-A594, M1-E593, M1-R592, M1-H591, M1-G590, M1-V589, M1-C588,M1-Q587, M1-N586, M1-P585, M1-Q584, M1-G583, M1-R582, M1-P581, M1-R580,M1-L579, M1-V578, M1-P577, M1-P576, M1-K575, M1-H574, M1-T573, M1-G572,M1-L571, M1-D570, M1-E569, M1-V568, M1-E567, M1-W566, M1-H565, M1-S564,M1-S563, M1-C562, M1-G561, M1-T560, M1-L559, M1-V558, M1-H557, M1-G556,M1-Q555, M1-Q554, M1-H553, M1-C552, M1-H551, M1-V550, M1-R549, M1-T548,M1-G547, M1-M546, M1-S545, M1-A544, M1-E543, M1-A542, M1-P541, M1-P540,M1-A539, M1-T538, M1-H537, M1-V536, M1-S535, M1-C534, M1-N533, M1-A532,M1-Q531, M1-P530, M1-L529, M1-L528, M1-C527, M1-C526, M1-R525, M1-A524,M1-I523, M1-A522, M1-Y521, M1-V520, M1-G519, M1-E518, M1-G517, M1-G516,M1-F515, M1-A514, M1-N513, M1-H512, M1-A511, M1-R510, M1-C509, M1-V508,M1-L507, M1-K506, M1-G505, M1-G504, M1-Q503, M1-A502, M1-E501, M1-M500,M1-R499, M1-E498, M1-G497, M1-R496, M1-R495, M1-K494, M1-G493, M1-S492,M1-R491, M1-S490, M1-F489, M1-S488, M1-S487, M1-C486, M1-S485, M1-L484,M1-L483, M1-E482, M1-E481, M1-D480, M1-P479, M1-A478, M1-C477, M1-R476,M1-A475, M1-V474, M1-A473, M1-T472, M1-A471, M1-M470, M1-R469, M1-T468,M1-P467, M1-G466, M1-S465, M1-H464, M1-A463, M1-S462, M1-W461, M1-V460,M1-T459, M1-R458, M1-C457, M1-F456, M1-1L455, M1-Q454, M1-W453, M1-G452,M1-A451, M1-G450, M1-H449, M1-T448, M1-S447, M1-P446, M1-P445, M1-1L444,M1-A443, M1-A442, M1-V441, M1-L440, M1-N439, M1-P438, M1-T437, M1-L436,M1-V435, M1-R434, M1-Q433, M1-D432, M1-E431, M1-P430, M1-F429, M1-W428,M1-A427, M1-E426, M1-N425, M1-I424, M1-V423, M1-D422, M1-K421, M1-A420,M1-S419, M1-F418, M1-H417, M1-I416, M1-L415, M1-R414, M1-Q413, M1-R412,M1411, M1-E410, M1-A409, M1-1L408, M1-T407, M1-L406, M1-E405, M1-P404,M1-E403, M1-A402, M1-S401, M1-L400, M1-M399, M1-M398, M1-A397, M1-A396,M1-I395, M1-G394, M1-A393, M1-V392, M1-H391, M1-A390, M1-A389, M1-A388,M1-Q387, M1-S386, M1-T385, M1-G384, M1-S383, M1-Q382, M1-S381, M1-V380,M1-F379, M1-C378, M1-T377, M1-S376, M1-C375, M1-D374, M1-S373, M1-S372,M1-A371, M1-G370, M1-I369, M1-I368, M1-D367, M1-E366, M1-G365, M1-P364,M1-A363, M1-F362, M1-L361, M1-D360, M1-V359, M1-C358, M1-R357, M1-G356,M1-F355, M1-N354, M1-T353, M1-G352, M1-L351, M1-T350, M1-G349, M1-L348,M1-T347, M1-V346, M1-P345, M1-Q344, M1-D343, M1-Q342, M1-A341, M1-N340,M1-T339, M1-A338, M1-G337, M1-V336, M1-T335, M1-I334, M1-V333, M1-E332,M1-P331, M1-A330, M1-S329, M1-A328, M1-P327, M1-S326, M1-Y325, M1-L324,M1-C323, M1-A322, M1-D321, M1-D320, M1-R319, M1-F318, M1-N317, M1-G316,M1-A315, M1-A314, M1-T313, M1-V312, M1-L311, M1-V310, M1-V309, M1-G308,M1-A307, M1-R306, M1-A305, M1-L304, M1-R303, M1-Q302, M1-C301, M1-A300,M1-A299, M1-N298, M1-L297, M1-V296, M1-R295, M1-S294, M1-Y293, M1-G292,M1-G291, M1-A290, M1-L289, M1-P288, M1-L287, M1-L286, M1-V285, M1-V284,M1-L283, M1-P282, M1-G281, M1-V280, M1-P279, M1-Q278, M1-V277, M1-L276,M1-Q275, M1-S274, M1-K273, M1-R272, M1-I271, M1-F270, M1-E269, M1-L268,M1-G267, M1-I266, M1-L265, M1-T264, M1-G263, M1-S262, M1-V261, M1-T260,M1-G259, M1-K258, M1-G257, M1-Q256, M1-C255, M1-N254, M1-L253, M1-V252,M1-R251, M1-L250, M1-S249, M1-R248, M1-M247, M1-S246, M1-A245, M1-G244,M1-K243, M1-A242, M1-V241, M1-G240, M1-A239, M1-D238, M1-R237, M1-G236,M1-S235, M1-V234, M1-V233, M1-G232, M1-A231, M1-L230, M1-H229, M1-T228,M1-G227, M1-H226, M1-S225, M1-D224, M1-C223, M1-K222, M1-S221, M1-A220,M1-Q219, M1-R218, M1-H217, M1-F216, M1-R215, M1-T214, M1-G213, M1-D212,M1-E211, M1-E210, M1-P209, M1-V208, M1-N207, M1-E206, M1-F205, M1-D204,M1-T203, M1-V202, M1-M201, M1-V200, M1-R199, M1-G198, M1-E197, M1-I196,M1-E195, M1-R194, M1-H193, M1-D192, M1-S191, M1-Q190, M1-I189, M1-S188,M1-T187, M1-D186, M1-L185, M1-L184, M1-Y183, M1-V182, M1-E181, M1-V180,M1-L179, M1-S178, M1-G177, M1-G176, M1-D175, M1-P174, M1-P173, M1-Q172,M1-Y171, M1-E170, M1-D169, M1-A168, M1-R167, M1-Y166, M1-R165, M1-P164,M1-P163, M1-T162, M1-I161, M1-R160, M1-E159, M1-L158, M1-N157, M1-W156,M1-P155, M1-I154, M1-S153, M1-Q152, M1-A151, M1-F50, M1-V149, M1-S148,M1-S147, M1-D146, M1-E145, M1-E144, M1-I143, M1-Y142, M1-D141, M1-V140,M1-H139, M1-P138, M1-L137, M1-K136, M1-L135, M1-A134, M1-L133, M1-E132,M1-L131, M1-L130, M1-D129, M1-G128, M1-S127, M1-M126, M1-K125, M1-V124,M1-L123, M1-F122, M1-G121, M1-P120, M1-L119, M1-L118, M1-G117, M1-H116,M1-F115, M1-V114, M1-H113, M1-L112, M1-I111, M1-K110, M1-T109, M1-L108,M1-Y107, M1-G106, M1-R105, M1-R104, M1-A103, M1-A102, M1-Q101, M1-A100,M1-Q99, M1-L98, M1-R97, M1-R96, M1-A95, M1-T94, M1-R93, M1-E92, M1-S91,M1-Q90, M1-S89, M1-L88, M1-H87, M1-T86, M1-E85, M1-E84, M1-K83, M1-L82,M1-V81, M1-V80, M1-V79, M1-Y78, M1-T77, M1-G76, M1-P75, M1-L74, M1-R73,M1-W72, M1-P71, M1-D70, M1-K69, M1-A68, M1-C67, M1-R66, M1-H65, M1-F64,M1-T63, M1-A62, M1-T61, M1-T60, M1-G59, M1-H58, M1-E57, M1-P56, M1-ASS,M1-E54, M1-A53, M1-L52, M1-G51, M1-D50, M1-E49, M1-E48, M1-S47, M1-R46,M1-L45, M1-A44, M1-L43, M1-V42, M1-L41, M1-E40, M1-E39, M1-Y38, M1-D37,M1-G36, M1-D35, M1-E34, M1-D33, M1-E32, M1-Q31, M1-A30, M1-R29, M1-A28,M1-G27, M1-A26, M1-P25, M1-G24, M1-L23, M1-L22, M1-L21, M1-L20, M1-L19,M1-L18, M1-L17, M1-L16, M1-L15, M1-P14, M1-L13, M1-P12, M1-W11, M1-W10,M1-S9, M1-R8, and/or M1-R7 of SEQ ID NO:5. Polypeptide sequences encodedby these polynucleotides are also provided as SEQ ID NO:38. In addition,the invention also encompasses polynucleotides encoding a polypeptidethat is at least as long as any one of the aforementioned polypeptides.The present invention also encompasses the use of these C-terminalPCSK9c deletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

TABLE I Total AA ATCC ® NT NT 5′ NT Seq Total Deposit SEQ Seq of Start3′ NT ID AA Gene CDNA No. Z and ID. of Codon of No. of No. CloneID DateVector No. X Clone of ORF ORF Y ORF 1. PCSK9b (also PTA-7622 pSPORT2 13175 250 1194 2 315 referred to as May 10, 2006 PCSK9-b) 2. PCSK9c (alsoPTA-7622 pSPORT1 3 3756 881 2449 4 523 referred to as May 10, 2006PCSK9-c)

Table I summarizes the information corresponding to each “Gene No.”described above. The nucleotide sequence identified as “NT SEQ ID NO:X”was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table I and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the correspondingdeposit number listed in “ATCC® Deposit No:Z and Date.” “Vector” refersto the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides inthe clone contig identified by “Gene No.” The deposited clone maycontain all or most of the sequence of SEQ ID NO:X. The nucleotideposition of SEQ ID NO:X of the putative start codon (methionine) isidentified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, isidentified as “AA SEQ ID NO:Y” although other reading frames can also beeasily translated using known molecular biology techniques. Thepolypeptides produced by these alternative open reading frames arespecifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ IDNO:Y is identified as “Total AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:X is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:Xor the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:Y may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a cDNA of theinvention deposited with the ATCC®, as set forth in Table I. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedcDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can beisolated in accordance with known methods using the sequence informationdisclosed herein. Such methods include preparing probes or primers fromthe disclosed sequence and identifying or amplifying the correspondinggene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelicvariants, and/or orthologs. The skilled artisan could, using procedureswell-known in the art, obtain the polynucleotide sequence correspondingto full-length genes (including, but not limited to the full-lengthcoding region), allelic variants, splice variants, orthologs, and/orspecies homologues of genes corresponding to SEQ ID NO:X, SEQ ID NO:Y,or a deposited clone, relying on the sequence from the sequencesdisclosed herein or the clones deposited with the ATCC®. For example,allelic variants and/or species homologues may be isolated andidentified by making suitable probes or primers which correspond to the5′, 3′, or internal regions of the sequences provided herein andscreening a suitable nucleic acid source for allelic variants and/or thedesired homologue.

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the protein, or may be a part ofa larger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:X,and/or a cDNA provided in ATCC® Deposit No. Z: The present inventionalso provides a polypeptide comprising, or alternatively consisting of,the sequence identified as SEQ ID NO:Y, and/or a polypeptide encoded bythe cDNA provided in ATCC® Deposit NO:Z. The present invention alsoprovides polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:Y,and/or a polypeptide sequence encoded by the cDNA contained in ATCC®Deposit No:Z.

Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:X, and/or a cDNA provided in ATCC® Deposit No.: that is lessthan, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:X, the sequence contained in a deposit, and/orthe nucleic acid sequence encoding the sequence disclosed as SEQ IDNO:Y.

The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystringent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table II below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R.

TABLE II Poly- Stringency nucleotide Hybrid Hybridization Temperatureand Wash Temp. Condition Hybrid± Length (bp) ‡ Buffer † and Buffer † ADNA:DNA > or equal to 65° C.; 1xSSC -or- 65° C.; 0.3xSSC 50 42° C.;1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC C DNA:RNA > orequal to 67° C.; 1xSSC -or- 67° C.; 0.3xSSC 50 45° C.; 1xSSC, 50%formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > or equal to70° C.; 1xSSC -or- 70° C.; 0.3xSSC 50 50° C.; 1xSSC, 50% formamide FRNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 65° C.; 4xSSC-or- 65° C.; 1xSSC 50 45° C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*;4xSSC Th*; 4xSSC I DNA:RNA > or equal to 67° C.; 4xSSC -or- 67° C.;1xSSC 50 45° C.; 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*;4xSSC K RNA:RNA > or equal to 70° C.; 4xSSC -or- 67° C.; 1xSSC 50 40°C.; 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M DNA:DNA >or equal to 50° C.; 4xSSC -or- 50° C.; 2xSSC 50 40° C.; 6xSSC, 50%formamide N DNA:DNA <50 Tn*; 6xSSC Tn*; 6xSSC O DNA:RNA > or equal to55° C.; 4xSSC -or- 55° C.; 2xSSC 50 42° C.; 6xSSC, 50% formamide PDNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 60° C.; 4xSSC-or- 60° C.; 2xSSC 50 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*;4xSSC Tr*; 4xSSC ‡ - The “hybrid length” is the anticipated for thehybridized region(s) of the hybridizing polynucleotides. Whenhybridizing a polynucleotide of unknown sequence, the hybrid is assumedto be that of the hybridizing polynucleotide of the present invention.When polynucleotides of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal sequence complementarity.Methods of aligning two or more polynucleotide sequences and/ordetermining the percent identity between two polynucleotide sequencesare well known in the art (e.g., MegAlign program of the DNASTAR ® suiteof programs, etc). † - SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M NaCland 15 mM sodium citrate) in the hybridization and wash buffers; washesare performed for 15 minutes after hybridization is complete. Thehydridizations and washes may additionally include 5X Denhardt'sreagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA,0.5% sodium pyrophosphate, and up to 50% formamide. *Tb-Tr: Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature Tm of the hybrids there Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log₁₀[Na+]) + 0.41(%G + C) − (600/N), where N is the number of bases in the hybrid, and[Na+] is the concentration of sodium ions in the hybridization buffer([Na+] for 1xSSC = .165M). ±The present invention encompasses thesubstitution of any one, or more DNA or RNA hybrid partners with eithera PNA, or a modified polynucleotide. Such modified polynucleotides areknown in the art and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequenceidentity (more preferably, at least 80% identity; and most preferably atleast 90%/0 or 95% identity) with the polynucleotide of the presentinvention to which they hybridize, where sequence identity is determinedby comparing the sequences of the hybridizing polynucleotides whenaligned so as to maximize overlap and identity while minimizing sequencegaps. The determination of identity is well known in the art, anddiscussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to thepolynucleotide sequences of the present invention, the clone depositedwith the ATCC®, and/or the cDNA encoding the polypeptides of the presentinvention. PCR techniques for the amplification of nucleic acids aredescribed in U.S. Pat. No. 4,683,195 and Saiki et al., Science,239:487-491 (1988). PCR, for example, may include the following steps,of denaturation of template nucleic acid (if double-stranded), annealingof primer to target, and polymerization. The nucleic acid probed or usedas a template in the amplification reaction may be genomic DNA, cDNA,RNA, or a PNA. PCR may be used to amplify specific sequences fromgenomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA.References for the general use of PCR techniques, including specificmethod parameters, include Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, StocktonPress, N Y, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and“PCR Protocols, A Guide to Methods and Applications”, Eds., Innis etal., Academic Press, New York, (1990).

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants,orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQID NO:X, the complementary strand thereto, and/or the cDNA sequencecontained in the deposited clone.

The present invention also encompasses variants of the polypeptidesequence, and/or fragments therein, disclosed in SEQ ID NO:Y, apolypeptide encoded by the polynucleotide sequence in SEQ ID NO:X,and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a PCSK9b or PCSK9c polypeptide having anamino acid sequence as shown in the sequence listing and described inSEQ ID NO:2 or SEQ ID NO:4 or the cDNA contained in ATCC® deposit No:Z;(b) a nucleotide sequence encoding a mature PCSK9b or PCSK9c polypeptidehaving the amino acid sequence as shown in the sequence listing anddescribed in SEQ ID NO:2 or SEQ ID NO:4 or the cDNA contained in ATCC®deposit No:Z; (c) a nucleotide sequence encoding a biologically activefragment of a PCSK9b or PCSK9c polypeptide having an amino acid sequenceshown in the sequence listing and described in SEQ ID NO:2 or SEQ IDNO:4 or the cDNA contained in ATCC® deposit No:Z; (d) a nucleotidesequence encoding an antigenic fragment of a PCSK9b or PCSK9cpolypeptide having an amino acid sequence sown in the sequence listingand described in SEQ ID NO:2 or SEQ ID NO:4 or the cDNA contained inATCC® deposit No:Z; (e) a nucleotide sequence encoding a PCSK9b orPCSK9c polypeptide comprising the complete amino acid sequence encodedby a human cDNA plasmid contained in SEQ ID NO:2 or SEQ ID NO:4 or thecDNA contained in ATCC® deposit No:Z; (f) a nucleotide sequence encodinga mature PCSK9b or PCSK9c polypeptide having an amino acid sequenceencoded by a human cDNA plasmid contained in SEQ ID NO:2 or SEQ ID NO:4or the cDNA contained in ATCC® deposit No:Z; (g) a nucleotide sequenceencoding a biologically active fragment of a PCSK9b or PCSK9cpolypeptide having an amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:2 or SEQ ID NO:4 or the cDNA contained inATCC® deposit No:Z; (h) a nucleotide sequence encoding an antigenicfragment of a PCSK9b or PCSK9c polypeptide having an amino acid sequenceencoded by a human cDNA plasmid contained in SEQ ID NO:X or the cDNAcontained in ATCC® deposit No:Z; and (i) a nucleotide sequencecomplimentary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences whichcomprise, or alternatively consist of, a polynucleotide sequence whichis at least about 80%, 85%, 90%, 91%, 92%, 93%, 93.6%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9% identical to, for example, any of the nucleotide sequences in(a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotidesencoded by these nucleic acid molecules are also encompassed by theinvention. In another embodiment, the invention encompasses nucleic acidmolecules which comprise, or alternatively, consist of a polynucleotidewhich hybridizes under stringent conditions, or alternatively, underlower stringency conditions, to a polynucleotide in (a), (b), (c), (d),(e), (f), (g), or (h), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolypeptides.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a PCSK9b or PCSK9c polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (b) a nucleotide sequence encoding a mature PCSK9b or PCSK9cpolypeptide having the amino acid sequence as shown in the sequencelisting and descried in Table I; (c) a nucleotide sequence encoding abiologically active fragment of a PCSK9b or PCSK9c polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (d) a nucleotide sequence encoding an antigenic fragment of aPCSK9b or PCSK9c polypeptide having an amino acid sequence as shown inthe sequence listing and descried in Table I; (e) a nucleotide sequenceencoding a PCSK9b or PCSK9c polypeptide comprising the complete aminoacid sequence encoded by a human cDNA in a cDNA plasmid contained in theATCC® Deposit and described in Table I; (f) a nucleotide sequenceencoding a mature PCSK9b or PCSK9c polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in theATCC® Deposit and described in Table I: (g) a nucleotide sequenceencoding a biologically active fragment of a PCSK9b or PCSK9cpolypeptide having an amino acid sequence encoded by a human cDNA in acDNA plasmid contained in the ATCC® Deposit and described in Table I;(h) a nucleotide sequence encoding an antigenic fragment of a PCSK9b orPCSK9c polypeptide having an amino acid sequence encoded by a human cDNAin a cDNA plasmid contained in the ATCC® deposit and described in TableI; and (i) a nucleotide sequence complimentary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively, consist of, a nucleotide sequence which isat least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%/0identical to, for example, any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to,the following non-limited examples, the polypeptide sequence identifiedas SEQ ID NO:Y, the polypeptide sequence encoded by a cDNA provided inthe deposited clone, and/or polypeptide fragments of any of thepolypeptides provided herein. Polynucleotides encoded by these nucleicacid molecules are also encompassed by the invention. In anotherembodiment, the invention encompasses nucleic acid molecules whichcomprise, or alternatively, consist of a polynucleotide which hybridizesunder stringent conditions, or alternatively, under lower stringencyconditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or(h), above. Polynucleotides which hybridize to the complement of thesenucleic acid molecules under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention, as are polypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to,for example, the polypeptide sequence shown in SEQ ID NO:Y, apolypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X,a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/orpolypeptide fragments of any of these polypeptides (e.g., thosefragments described herein). Polynucleotides which hybridize to thecomplement of the nucleic acid molecules encoding these polypeptidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompasses by the present invention, asare the polypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence referenced inTable I, the ORF (open reading frame), or any fragment specified asdescribed herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1/%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9%/0 identical to a nucleotide sequence of the presentinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multiple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the ALIGNX® softwareprogram (VECTOR NTI® suite of programs, version 6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the CLUSTALW alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

In addition to the above method of aligning two or more polynucleotideor polypeptide sequences to arrive at a percent identity value for thealigned sequences, it may be desirable in some circumstances to use amodified version of the CLUSTALW algorithm which takes into accountknown structural features of the sequences to be aligned, such as forexample, the SWISS-PROT® designations for each sequence. The result ofsuch a modified CLUSTALW algorithm may provide a more accurate value ofthe percent identity for two polynucleotide or polypeptide sequences.Support for such a modified version of CLUSTALW is provided within theCLUSTALW algorithm and would be readily appreciated to one of skill inthe art of bioinformatics.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the mRNA to those preferred by abacterial host such as E. coli).

Naturally occurring variants are called “allelic variants” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268:2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptideof the present invention may, in fact, result in a significant increasein one or more of the biological activities of the polypeptide(s). Forexample, biological activity of many polypeptides are governed by thepresence of regulatory domains at either one or both termini. Suchregulatory domains effectively inhibit the biological activity of suchpolypeptides in lieu of an activation event (e.g., binding to a cognateligand or receptor, phosphorylation, proteolytic processing, etc.).Thus, by eliminating the regulatory domain of a polypeptide, thepolypeptide may effectively be rendered biologically active in theabsence of an activation event.

Thus, the invention further includes polypeptide variants that showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved.

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thesame functions performed by the polypeptide of the present invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics (e.g.,chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thesame functions performed by the polypeptide of the present invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics (e.g.,chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gin, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in Table III below.

TABLE III For Amino Acid Code Replace with any of: Alanine A D-Ala, Gly,beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg,D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp,D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid ED-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro,D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine MD-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine FD-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,L-1-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylicacid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Informatics Computer Analysis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution ofamino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST®, CLUSTALW, GAP®, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the presentinvention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants createdthrough the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:X, the sequenceof the clone submitted in a deposit, and/or the cDNA encoding thepolypeptide disclosed as SEQ ID NO:Y. Such DNA Shuffling technology isknown in the art and more particularly described elsewhere herein (e.g.,WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples providedherein).

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of the present invention having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of thepolynucleotides of the invention, in addition to polypeptides encodedtherein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the cDNA in a deposited clone; is a portion of that shown in SEQ IDNO:X or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength, or at least about 875 nt in length, or at least about 837 nt inlength, or at least about 903 nt in length, or at least about 915 nt inlength, or at least about 930 nt in length, or at least about 945 nt inlength, or at least about 1000 nt in length, or at least about 1050 ntin length, or at least about 1100 nt in length, or at least about 1150nt in length, or at least about 1200 nt in length, or at least about1250 nt in length, or at least about 1300 nt in length, or at leastabout 1350 nt in length, or at least about 1400 nt in length, or atleast about 1450 nt in length, or at least about 1500 nt in length, orat least about 1554 nt in length. A fragment “at least 20 nt in length”for example, is intended to include 20 or more contiguous bases from thecDNA sequence contained in a deposited clone or the nucleotide sequenceshown in SEQ ID NO:X. In this context “about” includes the particularlyrecited value, a value larger or smaller by several (25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1) nucleotides, at either terminus, or at both termini. Thesenucleotide fragments have uses that include, but are not limited to, asdiagnostic probes and primers as discussed herein. Of course, largerfragments (e.g., 50, 150, 500, 600, 837, 903, 1554, 2000 nucleotides)are preferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, orthe complementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Also encompassed by the present invention are polynucleotideswhich hybridize to these nucleic acid molecules under stringenthybridization conditions or lower stringency conditions, as are thepolypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 279,301, 325, 350, 375, 400, 425, 450, 475, 500, or 518 amino acids inlength. In this context “about” includes the particularly recited rangesor values, and ranges or values larger or smaller by several (25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1) amino acids, at either extreme or at both extremes.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Preferred polypeptide fragments include the full-length protein. Furtherpreferred polypeptide fragments include the full-length protein having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of the full-lengthpolypeptide. Similarly, any number of amino acids, ranging from 1-30,can be deleted from the carboxy terminus of the full-length protein.Furthermore, any combination of the above amino and carboxy terminusdeletions are preferred. Similarly, polynucleotides encoding thesepolypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC® depositNo. Z or encoded by a polynucleotide that hybridizes to the complementof the sequence of SEQ ID NO:X or contained in ATCC® deposit No. Z understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNO:1 or SEQ ID NO:3), polynucleotide sequences of the complementarystrand of a polynucleotide sequence encoding an epitope of theinvention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined supra.

The term “epitopes” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length, or longer. Additional non-exclusive preferredantigenic epitopes include the antigenic epitopes disclosed herein, aswell as portions thereof. Antigenic epitopes are useful, for example, toraise antibodies, including monoclonal antibodies that specifically bindthe epitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:X and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,monovalent, bispecific, heteroconjugate, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. Moreover, the term “antibody” (Ab) or“monoclonal antibody” (Mab) is meant to include intact molecules, aswell as, antibody fragments (such as, for example, Fab and F(ab′)2fragments) which are capable of specifically binding to protein. Fab andF(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of the animal or plant, and may have lessnon-specific tissue binding than an intact antibody (Wahl et al., J.Nucl. Med . . . 24:316-325 (1983)). Thus, these fragments are preferred,as well as the products of a FAB or other immunoglobulin expressionlibrary. Moreover, antibodies of the present invention include chimeric,single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homologue of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologues of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M,5×10-6 M, 10-6 M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M,5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M,10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%/0, at least 75%, at least 70%, at least60%/0, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111 (Pt 2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9): 1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art.

The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988); and CurrentProtocols, Chapter 2; which are hereby incorporated herein by referencein its entirety). In a preferred method, a preparation of the PCSK9b orPCSK9c protein is prepared and purified to render it substantially freeof natural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity. For example, a polypeptide of the invention can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for the antigen. The administration ofthe polypeptides of the present invention may entail one or moreinjections of an immunizing agent and, if desired, an adjuvant. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art. For the purposes of the invention,“immunizing agent” may be defined as a polypeptide of the invention,including fragments, variants, and/or derivatives thereof, in additionto fusions with heterologous polypeptides and other forms of thepolypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections, thoughthey may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., pp. 563-681 (1981); Köhler et al., Eur. J.Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies includes, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a humanimmune system, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include polypeptides of the presentinvention or a fusion protein thereof. Preferably, the immunizing agentconsists of an PCSK9b or PCSK9c polypeptide or, more preferably, with aPCSK9b or PCSK9c polypeptide-expressing cell. Such cells may be culturedin any suitable tissue culture medium; however, it is preferable toculture cells in Earle's modified Eagle's medium supplemented with 10%fetal bovine serum (inactivated at about 56 degrees C.), andsupplemented with about 10 g/l of nonessential amino acids, about 1,000U/ml of penicillin, and about 100 ug/ml of streptomycin. Generally,either peripheral blood lymphocytes (“PBLs”) are used if cells of humanorigin are desired, or spleen cells or lymph node cells are used ifnon-human mammalian sources are desired. The lymphocytes are then fusedwith an immortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986), pp.59-103). Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. More preferred are the parent myeloma cellline (SP2O) as provided by the ATCC®. As inferred throughout thespecification, human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptides of the present invention. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra, and/or according to Wands et al. (Gastroenterology80:225-232 (1981)). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography.

The skilled artisan would acknowledge that a variety of methods exist inthe art for the production of monoclonal antibodies and thus, theinvention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples described herein. In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC®. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab)2 fragments of the inventionmay be produced by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab)2 fragments). F(ab′)2 fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabillyet al., Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985);U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which areincorporated herein by reference in their entirety. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of Cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Genpharm (SanJose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Such anti-idiotypic antibodies capable of binding to the PCSK9b orPCSK9c polypeptide can be produced in a two-step procedure. Such amethod makes use of the fact that antibodies are themselves antigens,and therefore, it is possible to obtain an antibody that binds to asecond antibody. In accordance with this method, protein specificantibodies are used to immunize an animal, preferably a mouse. Thesplenocytes of such an animal are then used to produce hybridoma cells,and the hybridoma cells are screened to identify clones that produce anantibody whose ability to bind to the protein-specific antibody can beblocked by the polypeptide. Such antibodies comprise anti-idiotypicantibodies to the protein-specific antibody and can be used to immunizean animal to induce formation of further protein-specific antibodies.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, Preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards a polypeptide of the present invention, the othermay be for any other antigen, and preferably for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:4.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

More preferably, a clone encoding an antibody of the present inventionmay be obtained according to the method described in the Example sectionherein.

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIBTECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2 or SEQ ID NO:4 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2 or SEQ ID NO:4 may be fused or conjugated to the above antibodyportions to facilitate purification. One reported example describeschimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amrnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of syntheticantibodies directed against the polypeptides of the present invention.One example of synthetic antibodies is described in Radrizzani, M., etal., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class ofsynthetic antibodies has been described and are referred to asmolecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies,peptides, and enzymes are often used as molecular recognition elementsin chemical and biological sensors. However, their lack of stability andsignal transduction mechanisms limits their use as sensing devices.Molecularly imprinted polymers (MIPs) are capable of mimicking thefunction of biological receptors but with less stability constraints.Such polymers provide high sensitivity and selectivity while maintainingexcellent thermal and mechanical stability. MIPs have the ability tobind to small molecules and to target molecules such as organics andproteins' with equal or greater potency than that of natural antibodies.These “super” MIPs have higher affinities for their target and thusrequire lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementarysize, shape, charge and functional groups of the selected target byusing the target molecule itself (such as a polypeptide, antibody,etc.), or a substance having a very similar structure, as its “print” or“template.” MIPs can be derivatized with the same reagents afforded toantibodies. For example, fluorescent ‘super’ MIPs can be coated ontobeads or wells for use in highly sensitive separations or assays, or foruse in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of thepresent invention may be useful in screening for compounds that bind tothe polypeptide(s) of the invention. Such a MIP would serve the role ofa synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

MIPs have also been shown to be useful in “sensing” the presence of itsmimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron.,16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001)). For example, a MIP designed using a polypeptideof the present invention may be used in assays designed to identify, andpotentially quantitate, the level of said polypeptide in a sample. Sucha MIP may be used as a substitute for any component described in theassays, or kits, provided herein (e.g., ELISA, etc.).

A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. Forexample, such antibodies may be used in diagnostic assays to detect thepresence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp. 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219 (1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present inventionare useful for the affinity purification of such polypeptides fromrecombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a SEPHADEX® resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses of Antibodies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention areuseful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibodydirected against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced ina plant (e.g., cloning the gene of the antibody directed against apolypeptide of the present invention, and transforming a plant with asuitable vector comprising said gene for constitutive expression of theantibody within the plant), and the plant subsequently ingested by ananimal, thereby conferring temporary immunity to the animal for thespecific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferablypolyclonal antibodies, more preferably monoclonal antibodies, and mostpreferably single-chain antibodies, can be used as a means of inhibitinggene expression of a particular gene, or genes, in a human, mammal,and/or other organism. See, for example, International PublicationNumber WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. Theapplication of such methods for the antibodies of the present inventionare known in the art, and are more particularly described elsewhereherein.

In yet another embodiment, antibodies of the present invention may beuseful for multimerizing the polypeptides of the present invention. Forexample, certain proteins may confer enhanced biological activity whenpresent in a multimeric state (i.e., such enhanced activity may be dueto the increased effective concentration of such proteins whereby moreprotein is available in a localized location).

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, N Y (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E.W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging with Antibodies

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest; and (b) comparing the level ofgene expression with a standard gene expression level, whereby anincrease or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of aberrantexpression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest; and (b) comparing the level ofgene expression with a standard gene expression level, whereby anincrease or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: (a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; (b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level);(c) determining background level; and (d) detecting the labeled moleculein the subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because certain proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1,CH2, CH3, and any combination thereof, including both entire domains andportions thereof), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A 0 464 533 (Canadian counterpart 2,045,869) disclosesfusion proteins comprising various portions of the constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0 232 262.) Alternatively, deletingthe Fc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences (also referred to as “tags”). Due to the availabilityof antibodies specific to such “tags”, purification of the fusedpolypeptide of the invention, and/or its identification is significantlyfacilitated since antibodies specific to the polypeptides of theinvention are not required. Such purification may be in the form of anaffinity purification whereby an anti-tag antibody or another type ofaffinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

The skilled artisan would acknowledge the existence of other “tags”which could be readily substituted for the tags referred to supra forpurification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the FLAG®—peptide—i.e., the octapeptide sequence DYKDDDDK (SEQID NO:56), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci.USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to ninecodons encoding a repeating series of up to nine arginine amino acids tothe coding region of a polynucleotide of the present invention. Theinvention also encompasses chemically derivitizing a polypeptide of thepresent invention with a repeating series of up to nine arginine aminoacids. Such a tag, when attached to a polypeptide, has recently beenshown to serve as a universal pass, allowing compounds access to theinterior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention,including fragments and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be determined, toxins, such as ricin,saporin (Mashiba H, et al., Ann. N. Y. Acad. Sci. 1999; 886:233-5), orHC toxin (Tonukari N J, et al., Plant Cell. 2000 February;12(2):237-248), for example. Such fusions could be used to deliver thetoxins to desired tissues for which a ligand or a protein capable ofbinding to the polypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed againstpolypeptides of the present invention, including variants and fragmentsthereof, to said toxins for delivering the toxin to specific locationsin a cell, to specific tissues, and/or to specific species. Suchbifunctional antibodies are known in the art, though a review describingadditional advantageous fusions, including citations for methods ofproduction, can be found in P. J. Hudson, Curr. Opp. In. Imm.11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fungal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC®Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; PBLUESCRIPT® vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express thepolypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O2. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O2. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination,resulting in the formation of a new transcription unit (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761,issued Mar. 31, 1998; International Publication No. WO 96/29411,published Sep. 26, 1996; International Publication No. WO 94/12650,published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG®, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N- or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about1 kDa and about 100 kDa (the term “about” indicating that inpreparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated thatthe polymeric residues may contain functional groups in addition, forexample, to those typically involved in linking the polymeric residuesto the polypeptides of the present invention. Such functionalitiesinclude, for example, carboxyl, amine, hydroxy and thiol groups. Thesefunctional groups on the polymeric residues can be further reacted, ifdesired, with materials that are generally reactive with such functionalgroups and which can assist in targeting specific tissues in the bodyincluding, for example, diseased tissue. Exemplary materials which canbe reacted with the additional functional groups include, for example,proteins, including antibodies, carbohydrates, peptides, glycopeptides,glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used toderivatize the polypeptides of the present invention can be a saccharideresidue. Exemplary saccharides which can be derived include, forexample, monosaccharides or sugar alcohols, such as erythrose, threose,ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol andsedoheptulose, with preferred monosaccharides being fructose, mannose,xylose, arabinose, mannitol and sorbitol; and disaccharides, such aslactose, sucrose, maltose and cellobiose. Other saccharides include, forexample, inositol and ganglioside head groups. Other suitablesaccharides, in addition to those exemplified above, will be readilyapparent to one skilled in the art based on the present disclosure.Generally, saccharides which may be used for derivitization includesaccharides that can be attached to the polypeptides of the inventionvia alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

The invention encompasses derivitization of the polypeptides of thepresent invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof Accordingly, suitable polymers include, for example,proteins, such as albumin, polyalginates, and polylactide-coglycolidepolymers. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as PLURONIC®, commercially available from BASF, Parsippany,N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes(such as, for example, polypropylene glycol), polyurethanes (such as,for example, polyvinyl alcohol (PVA), polyvinyl chloride andpolyvinylpyrrolidone), polyamides including nylon, polystyrene,polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbonpolymers (such as, for example, polytetrafluoroethylene), acrylate,methacrylate, and polymethylmethacrylate, and derivatives thereof.Methods for the preparation of derivatized polypeptides of the inventionwhich employ polymers as stabilizing compounds will be readily apparentto one skilled in the art, in view of the present disclosure, whencoupled with information known in the art, such as that described andreferred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of whichis hereby incorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of thepolypeptides of the present invention. Such additional modifications areknown in the art, and are specifically provided, in addition to methodsof derivitization, etc., in U.S. Pat. No. 6,028,066, which is herebyincorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone (including fragments,variants, splice variants, and fusion proteins, corresponding to thesepolypeptides as described herein). These homomers may containpolypeptides having identical or different amino acid sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly polypeptides having an identical amino acid sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingpolypeptides having different amino acid sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing polypeptides having identical or different amino acidsequences) or a homotrimer (e.g., containing polypeptides havingidentical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,osteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between FLAG® polypeptide sequence contained in fusionproteins of the invention containing FLAG® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in FLAG® fusion proteins of the invention and anti-FLAG®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could beoperatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).Preferred polynucleotides correspond to the noncoding regions of thecDNAs because the coding sequences are more likely conserved within genefamilies, thus increasing the chance of cross hybridization duringchromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedorganisms as compared to unaffected organisms can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anorganism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

By “measuring the expression level of a polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman organism, body fluids, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as the followingnon-limiting examples, sputum, amniotic fluid, urine, saliva, breastmilk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.)which contain the polypeptide of the present invention, and other tissuesources found to express the polypeptide of the present invention.Methods for obtaining tissue biopsies and body fluids from organisms arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

The method(s) provided above may preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supraare hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 40-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Antisense techniques are discussed, for example, in Okano, J. Neurochem.56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). Both methods rely on binding of thepolynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

The present invention encompasses the addition of a nuclear localizationsignal, operably linked to the 5′ end, 3′ end, or any location therein,to any of the oligonucleotides, antisense oligonucleotides, triple helixoligonucleotides, ribozymes, PNA oligonucleotides, and/orpolynucleotides, of the present invention. See, for example, G. Cutrona,et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporatedherein by reference.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an organisms genome. These sequences can be usedto prepare PCR primers for amplifying and isolating such selected DNA,which can then be sequenced. Using this technique, organisms can beidentified because each organism will have a unique set of DNAsequences. Once an unique ID database is established for an organism,positive identification of that organism, living or dead, can be madefrom extremely small tissue samples. Similarly, polynucleotides of thepresent invention can be used as polymorphic markers, in addition to,the identification of transformed or non-transformed cells and/ortissues.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, when presented withtissue of unknown origin. Appropriate reagents can comprise, forexample, DNA probes or primers specific to particular tissue preparedfrom the sequences of the present invention. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.Moreover, as mentioned above, such reagents can be used to screen and/oridentify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific mRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteinscan also be detected in vivo by imaging. Antibody labels or markers forin vivo imaging of protein include those detectable by X-radiography,NMR or ESR. For X-radiography, suitable labels include radioisotopessuch as barium or cesium, which emit detectable radiation but are notovertly harmful to the subject. Suitable markers for NMR and ESR includethose with a detectable characteristic spin, such as deuterium, whichmay be incorporated into the antibody by labeling of nutrients for therelevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of a polypeptide of the presentinvention in cells or body fluid of an individual; and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Moreover, polypeptides of the present invention can be used to treat,prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat, prevent, and/or diagnose disease. Forexample, administration of an antibody directed to a polypeptide of thepresent invention can bind and reduce overproduction of the polypeptide.Similarly, administration of an antibody can activate the polypeptide,such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofa polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the invention thatoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO 90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the polynucleotides of theinvention can also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

The polynucleotide vector constructs of the invention used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter, or heterologous promoters, such as thecytomegalovirus (CMV) promoter, the respiratory syncytial virus (RSV)promoter, inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter,the ApoAI promoter, human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter,retroviral LTRs; the b-actin promoter, and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs of the inventionare complexed in a liposome preparation. Liposomal preparations for usein the instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA,84:7413-7416 (1987), which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), whichis herein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark LIPOFECTIN®,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad.Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingpolypeptides of the invention. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO4 precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding polypeptides of the invention.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotides of the invention contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartz et al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ads, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotides encoding polypeptides of the present invention maybe administered along with other polynucleotides encoding angiogenicproteins. Angiogenic proteins include, but are not limited to, acidicand basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3(VEGF-B), epidermal growth factor alpha and beta, platelet-derivedendothelial cell growth factor, platelet-derived growth factor, tumornecrosis factor alpha, hepatocyte growth factor, insulin like growthfactor, colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

Preferably, the polynucleotide encoding a polypeptide of the inventioncontains a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., ALZA® minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers. (Kaneda et al.,Science, 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian. Therapeuticcompositions of the present invention can be administered to any animal,preferably to mammals and birds. Preferred mammals include humans, dogs,cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humansbeing particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention could be used to treat or prevent blood coagulationdiseases, disorders, and/or conditions (e.g., afibrinogenemia, factordeficiencies, arterial thrombosis, venous thrombosis, etc.), bloodplatelet diseases, disorders, and/or conditions (e.g. thrombocytopenia),or wounds resulting from trauma, surgery, or other causes.Alternatively, a polynucleotides or polypeptides, or agonists orantagonists of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention are may also be useful for the detection, prognosis,treatment, and/or prevention of heart attacks (infarction), strokes,scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation,uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

Examples of autoimmune diseases, disorders, and/or conditions that canbe treated, prevented, and/or diagnosed or detected by the presentinvention include, but are not limited to: Addison's Disease, hemolyticanemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis,allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome,Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis,Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies,Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be used to treat, prevent, and/or diagnoseorgan rejection or graft-versus-host disease (GVHD). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention may also be used to modulate inflammation. Forexample, the polypeptide or polynucleotide or agonists or antagonist mayinhibit the proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat, prevent,and/or diagnose inflammatory conditions, both chronic and acuteconditions, including chronic prostatitis, granulomatous prostatitis andmalacoplakia, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of theinvention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present inventioninclude, but are not limited to neoplasms located in the: colon,abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventingcell proliferative diseases, disorders, and/or conditions by insertinginto an abnormally proliferating cell a polynucleotide of the presentinvention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating,preventing, and/or diagnosing one or more of the described diseases,disorders, and/or conditions. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating, preventing, and/or diagnosing asubject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of diseases, disorders, and/orconditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses. 50(5):423-33 (1998), Chem. Biol. Interact. April24; 111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such therapeuticaffects of the present invention may be achieved either alone, or incombination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used to treat, prevent, and/or diagnose cardiovasculardiseases, disorders, and/or conditions, including peripheral arterydisease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cor triatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of theinvention, are especially effective for the treatment of critical limbischemia and coronary disease.

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides of the invention may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringpolynucleotides of the invention are described in more detail herein.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can betreated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of thepresent invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used totreat, prevent,and/or diagnose any of these symptoms or diseases. In specificembodiments, polynucleotides, polypeptides, or agonists or antagonistsof the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention can be used to differentiate, proliferate, and attractcells, leading to the regeneration of tissues. (See, Science 276:59-87(1997).) The regeneration of tissues could be used to repair, replace,or protect tissue damaged by congenital defects, trauma (wounds, burns,incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonistof the present invention may increase regeneration of tissues difficultto heal. For example, increased tendon/ligament regeneration wouldquicken recovery time after damage. A polynucleotide or polypeptideand/or agonist or antagonist of the present invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated, prevented, and/or diagnosed include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention to proliferate and differentiate nerve cells. Diseasesthat could be treated, prevented, and/or diagnosed using this methodinclude central and peripheral nervous system diseases, neuropathies, ormechanical and traumatic diseases, disorders, and/or conditions (e.g.,spinal cord disorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated, prevented, and/or diagnosed using the polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2): Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention bindscan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS® sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptides of the invention. Biologically active fragments are thoseexhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and 3[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (c) determining if a biological activity of thepolypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the inventionexperimentally by using the beta-pleated sheet regions contained in thepolypeptide sequence of the protein. Accordingly, specific embodimentsof the invention are directed to polynucleotides encoding polypeptideswhich comprise, or alternatively consist of, the amino acid sequence ofeach beta pleated sheet regions in a disclosed polypeptide sequence.Additional embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, anycombination or all of contained in the polypeptide sequences of theinvention. Additional preferred embodiments of the invention aredirected to polypeptides which comprise, or alternatively consist of,the amino acid sequence of each of the beta pleated sheet regions in oneof the polypeptide sequences of the invention. Additional embodiments ofthe invention are directed to polypeptides which comprise, oralternatively consist of, any combination or all of the beta pleatedsheet regions in one of the polypeptide sequences of the invention.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

The human PCSK9b or PCSK9c polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a PCSK9b or PCSK9cpolypeptide, or a bindable peptide fragment, of this invention,comprising providing a plurality of compounds, combining the PCSK9b orPCSK9c polypeptide, or a bindable peptide fragment, with each of aplurality of compounds for a time sufficient to allow binding undersuitable conditions and detecting binding of the PCSK9b or PCSK9cpolypeptide or peptide to each of the plurality of test compounds,thereby identifying the compounds that specifically bind to the PCSK9bor PCSK9c polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novelhuman PCSK9b or PCSK9c polypeptides and/or peptides are provided by thepresent invention and comprise combining a potential or candidatecompound or drug modulator of calpain biological activity with an PCSK9bor PCSK9c polypeptide or peptide, for example, the PCSK9b or PCSK9camino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4, andmeasuring an effect of the candidate compound or drug modulator on thebiological activity of the PCSK9b or PCSK9c polypeptide or peptide. Suchmeasurable effects include, for example, physical binding interaction;the ability to cleave a suitable calpain substrate; effects on nativeand cloned PCSK9b or PCSK9c-expressing cell line; and effects ofmodulators or other calpain-mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of the novel PCSK9b or PCSK9c polypeptides of the presentinvention comprises combining a potential or candidate compound or drugmodulator of a calpain biological activity with a host cell thatexpresses the PCSK9b or PCSK9c polypeptide and measuring an effect ofthe candidate compound or drug modulator on the biological activity ofthe PCSK9b or PCSK9c polypeptide. The host cell can also be capable ofbeing induced to express the PCSK9b or PCSK9c polypeptide, e.g., viainducible expression. Physiological effects of a given modulatorcandidate on the PCSK9b or PCSK9c polypeptide can also be measured.Thus, cellular assays for particular calpain modulators may be eitherdirect measurement or quantification of the physical biological activityof the PCSK9b or PCSK9c polypeptide, or they may be measurement orquantification of a physiological effect. Such methods preferably employa PCSK9b or PCSK9c polypeptide as described herein, or an overexpressedrecombinant PCSK9b or PCSK9c polypeptide in suitable host cellscontaining an expression vector as described herein, wherein the PCSK9bor PCSK9c polypeptide is expressed, overexpressed, or undergoesupregulated expression.

Another aspect of the present invention embraces a method of screeningfor a compound that is capable of modulating the biological activity ofa PCSK9b or PCSK9c polypeptide, comprising providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a PCSK9b or PCSK9c polypeptide, or a functional peptide orportion thereof (e.g., SEQ ID NO:2 or SEQ ID NO:4); determining thebiological activity of the expressed PCSK9b or PCSK9c polypeptide in theabsence of a modulator compound; contacting the cell with the modulatorcompound and determining the biological activity of the expressed PCSK9bor PCSK9c polypeptide in the presence of the modulator compound. In sucha method, a difference between the activity of the PCSK9b or PCSK9cpolypeptide in the presence of the modulator compound and in the absenceof the modulator compound indicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as calpain modulators can be any small chemicalcompound, or biological entity (e.g., protein, sugar, nucleic acid,lipid). Test compounds will typically be small chemical molecules andpeptides. Generally, the compounds used as potential modulators can bedissolved in aqueous or organic (e.g., DMSO-based) solutions. The assaysare designed to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned forthe detection of modulators of the novel PCSK9b or PCSK9cpolynucleotides and polypeptides described herein. Such high throughputscreening methods typically involve providing a combinatorial chemicalor peptide library containing a large number of potential therapeuticcompounds (e.g., ligand or modulator compounds). Such combinatorialchemical libraries or ligand libraries are then screened in one or moreassays to identify those library members (e.g., particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds so identified can serve as conventional lead compounds, orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara etal., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc.,114:9217-9218), analogous organic synthesis of small compound libraries(Chen et al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Choet al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbellet al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (seeAusubel, Berger and Sambrook, all supra), peptide nucleic acid libraries(U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al.,1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287),carbohydrate libraries (e.g., Liang et al., 1996, Science,274-1520-1522) and U.S. Pat. No. 5,593,853), small organic moleculelibraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; andU.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingan ion channel is attached to a solid phase substrate. In such highthroughput assays, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

In another of its aspects, the present invention encompasses screeningand small molecule (e.g., drug) detection assays which involve thedetection or identification of small molecules that can bind to a givenprotein, i.e., a PCSK9b or PCSK9c polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

To purify a PCSK9b or PCSK9c polypeptide or peptide to measure abiological binding or ligand binding activity, the source may be a wholecell lysate that can be prepared by successive freeze-thaw cycles (e.g.,one to three) in the presence of standard protease inhibitors. ThePCSK9b or PCSK9c polypeptide may be partially or completely purified bystandard protein purification methods, e.g., affinity chromatographyusing specific antibody described infra, or by ligands specific for anepitope tag engineered into the recombinant PCSK9b or PCSK9c polypeptidemolecule, also as described herein. Binding activity can then bemeasured as described.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe PCSK9b or PCSK9c polypeptides according to the present invention area preferred embodiment of this invention. It is contemplated that suchmodulatory compounds may be employed in treatment and therapeuticmethods for treating a condition that is mediated by the novel PCSK9b orPCSK9c polypeptides by administering to an individual in need of suchtreatment a therapeutically effective amount of the compound identifiedby the methods described herein.

In addition, the present invention provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by the PCSK9b or PCSK9c polypeptides of theinvention, comprising administering to the individual a therapeuticallyeffective amount of the PCSK9b or PCSK9c-modulating compound identifiedby a method provided herein.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:X, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoRI site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoRI/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

Antisense oligonucleotides may be single or double stranded. Doublestranded RNA's may be designed based upon the teachings of Paddison etal., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); and InternationalPublication Nos. WO 01/29058, and WO 99/32619; which are herebyincorporated herein by reference.

SiRNA reagents are specifically contemplated by the present invention.Such reagents are useful for inhibiting expression of thepolynucleotides of the present invention and may have therapeuticefficacy. Several methods are known in the art for the therapeutictreatment of disorders by the administration of siRNA reagents. One suchmethod is described by Tiscornia et al (PNAS, 100(4):1844-1848 (2003));WO0409769, filed Jul. 18^(th), 2003; and Reich S J et al., Mol Vis. 2003May 30; 9:210-6, which are incorporated by reference herein in itsentirety.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652(1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication NO: WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end,the oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequenceof the invention could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/ordiagnose the diseases described herein.

Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may increase the organisms ability, either directly orindirectly, to initiate and/or maintain biotic associations with otherorganisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the host organismsability, either directly or indirectly, to initiate and/or maintainbiotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

In an alternative embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may decrease the hostorganisms ability to form biotic associations with another organism,either directly or indirectly. The mechanism by which a polynucleotideor polypeptide and/or agonist or antagonist of the present invention maydecrease the host organisms ability, either directly or indirectly, toinitiate and/or maintain biotic associations with another organism isvariable, though may include, modulating osmolarity to undesirablelevels, modulating pH to undesirable levels, modulating secretions oforganic acids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the decreased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling and Communication”,eds., R. England, G. Hobbs, N. Bainton, and D. McL. Roberts, CambridgeUniversity Press, Cambridge, (1999); which is hereby incorporated hereinby reference).

The hosts ability to maintain biotic associations with a particularpathogen has significant implications for the overall health and fitnessof the host. For example, human hosts have symbiosis with entericbacteria in their gastrointestinal tracts, particularly in the small andlarge intestine. In fact, bacteria counts in feces of the distal colonoften approach 10¹² per milliliter of feces. Examples of bowel flora inthe gastrointestinal tract are members of the Enterobacteriaceae,Bacteroides, in addition to a-hemolytic streptococci, E. coli,Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, andyeasts. Such bacteria, among other things, assist the host in theassimilation of nutrients by breaking down food stuffs not typicallybroken down by the hosts digestive system, particularly in the hostsbowel. Therefore, increasing the hosts ability to maintain such a bioticassociation would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularlyhumans, has been associated with the following disorders: diarrhea,ileus, chronic inflammatory disease, bowel obstruction, duodenaldiverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

The composition of the intestinal flora, for example, is based upon avariety of factors, which include, but are not limited to, the age,race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

Although the predominate intestinal flora comprises anaerobic organisms,an underlying percentage represents aerobes (e.g., E. coli). This issignificant as such aerobes rapidly become the predominate organisms inintraabdominal infections—effectively becoming opportunistic early ininfection pathogenesis. As a result, there is an intrinsic need tocontrol aerobe populations, particularly for immune compromisedindividuals.

In a preferred embodiment, a polynucleotides and polypeptides, includingagonists, antagonists, and fragments thereof, are useful for inhibitingbiotic associations with specific enteric symbiont organisms in aneffort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, butalso on an in the integument. As opposed to the gastrointestinal flora,the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M. sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P. acnes), gram-negative bacilli (e.g.,Acinebacter species), and fungi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number ofsignificant diseases and/or disorders, which include, but are notlimited to the following: impetigo, ecthyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.,Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the organisms abilityto synthesize and/or release a pheromone. Such a pheromone may, forexample, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones. Preferably, any of the pheromones, and/or volatilesreleased from the organism, or induced, by a polynucleotide orpolypeptide and/or agonist or antagonist of the invention havebehavioral effects the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat, prevent, and/or diagnose neuronal damagewhich occurs in certain neuronal disorders or neuro-degenerativeconditions such as Alzheimer's disease, Parkinson's disease, andAIDS-related complex. The polypeptide of the invention may have theability to stimulate chondrocyte growth, therefore, they may be employedto enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

The polypeptides of the present invention may be employed to stimulategrowth and differentiation of hematopoietic cells and bone marrow cellswhen used in combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestrial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

Also preferred is a method of treatment of an individual in need of anincreased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books,GENBANK® Accession numbers, SWISS-PROT® Accession numbers, or otherdisclosures) in the Background of the Invention, Detailed Description,Brief Description of the Figures, and Examples is hereby incorporatedherein by reference in their entirety. Further, the hard copy of theSequence Listing submitted herewith, in addition to its correspondingComputer Readable Form, are incorporated herein by reference in theirentireties.

REFERENCES

-   Lusis, A J. Atherosclerosis. Nature. 407, 233-241 (2000).-   Fredrickson, D S., Levy, R I. & Lees, R S. N. Eng. J Med. 276,    273-281 (1967).-   Austin, M A., King, M C., Bawol, R D., Hulley, S B. & Friedman, G D.    Am. J Epidemiol. 125, 308-318 (1987).-   Perusse, L. Arteriosclerosis. 9, 308-318 (1989).-   Rice, T., Vogler, G P., Laskarzewski, P M., Perry, T S. & Rao, D C.    Hum. Biol. 63, 419-439 (1991).-   Khachadurian, A K., Am. J Med. 37, 402-407, (1964).-   Morganroth, J., Levy, R I., McMahon, A E. & Gotto, A M Jr. J    Pediatr. 85, 639-643 (1974).-   Goldstein, J L. & Brown, M S., Johns Hopkins Med. J 143, 8-16    (1978).-   Brown, M S. & Goldstein, J L. Proc. Natl. Acad. Sci. USA. 96,    11041-11048 (1999).-   Varret, M. et al. Am. J. Hum. Genet. 64, 1378-1387 (1999).-   Innerarity, T L. et al. Proc. Natl. Acad. Sci. USA. 84, 6919-6923    (1987).-   Hunt, S C. et al. Arterioscler. Thromb. Vasc. Biol. 20, 1089-1093    (2000).-   Horton J. D., Cohen J. C., Hobbs H. H. 2007. Molecular biology of    PCSK9: its role in LDL metabolism. Trends in Biochem. Sci. 332(2):    71-77.-   Berge, K. E., Ose L, Leren, T. P. 2006. Missense mutations in the    PCSK9 gene are associated with hypocholesterolemia and possibly    increased response to statin therapy. Arterioscl. Thromb. Vasc.    Biol. 26(5): 1094-1100.-   Zhao Z, Tuakli-Wosornu, Y, Lagace T. A., Kinch L., Grishin N. V.,    Horton J. D., Cohen J. C., Hobbs H. H. 2006. Molecular    characterization of loss-of-function mutations in PCSK and    identification of a compound heterozygote. Amer. J. Human Gen. 79:    514-523.-   Sun X.-M, Eden E. r., Tosi I., Neuwirth C. K., Wile D., Naoumova R.    P., Soutar A. K. 2005. Evidence for effect of mutant PCSK9 on    apolipoprotein B secretion as the cause of unusually severe dominant    hypercholesterolemia. Hum Mol. Gen. 14(9). 1161-1169.-   Maxwell K. N., Breslow J. L. 2004. Adenoviral-mediated expression of    Pcsk9 in mice results in a low-density lipoprotein receptor knockout    phenotype. Proc. Nat. Amer. Sci. 101: 7100-7105.-   Benjannet S, Rhainds, D., Essalmani R., Mayne J., Wickham L., Jin    W., Asselin M.-C., Hamelin J., Varret M., Allard D., Trillard M.,    Abifadel M., Tebon A., Attie A. D. Rader D. J., Boileau C.,    Brissette L., Chretien M., Prat A., Seidah N. G. 2004. NARC-1/PCSK9    and its natural mutants. J. Bio. Chem. 279: 48865-48875.

EXAMPLES Example 1 Method of Cloning the Novel Human PCSK9bPolynucleotide of the Present Invention

Using the sequence AK124635.1, a sense 80-mer oligo with biotin on the5′ end was designed with the following sequence:

(SEQ ID NO: 7) 5′bGGCGCTTTCACCAGTGGCTGGGATGTGCTCTGTAGTTTCTGTGTGTTAACTATAAGGTTGACTTTATGCTCATTCCCTCC-3′

One microliter (200 picograms) of the biotinylated oligo was added tosix microliters (six micrograms) of a anti-sense single-strandedcovalently closed circular brain cDNA library and seven microliters of100% formamide in a 0.2 ml PCR tube. The mixture was heated in a thermalcycler to 95° C. for 2 minutes. Fourteen microliters of 2× hybridizationbuffer (50% formamide, 1.5M NaCl, 0.04M NaPO₄ pH7.2, 5 mM EDTA, 0.2%SDS) were added to the heated probe/cDNA library mixture and incubatedat 42° C. for 24 hours. Hybrids between the biotinylated oligo and thecircular cDNA were isolated by diluting the hybridization mixture to 228microliters in a solution containing 1M NaCl, 10 mM Tris-HCl pH7.5, 1 mMEDTA, pH8.0 and adding 125 microliters of streptavidin magnetic beads.This solution was incubated at 42° C. for 60 minutes mixing every 5minutes to resuspend the beads. The beads were separated from thesolution with a magnet and washed three times in 200 microliters of0.1×SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinylatedoligo/streptavidin magnetic bead complex by adding 50 microliters of0.1N NaOH and incubating at room temperature for 10 minutes. Sixmicroliters of 3M Sodium Acetate were added along with 20 micrograms ofglycogen and the solution was ethanol precipitated with 120 microlitersof 100% ethanol. The DNA was resuspended in 12 microliters of TE (10 mMTris-HCl pH8.0, 1 mM EDTA pH8.0).

The single stranded cDNA was converted into double strands in a thermalcycler by mixing 5 microliters of the captured DNA with 1.5 microliters10 micromolar gene specific sense repair primer with the followingsequence: 5′-CTAGGCCTCCCTITCCTTGT-3′ (SEQ ID NO:13), and 1.5 microlitersof 10×PCR buffer. The mixture was heated to 95° C. for 20 seconds, thenramped down to 59° C. At this time 15 microliters of a repair mix, thatwas preheated to 70° C., was added (repair mix contains 0.5 microlitersof 10 mM dNTPs (2.5 mM each), 1.5 microliters of 10×PCR buffer, 12.75microliters of water, and 0.25 microliters or 1.25 U of Taq polymerase).The solution was ramped back to 73° C. and incubated for 23 minutes. Therepaired DNA was ethanol precipitated and resuspended in 10 microlitersof TE. Two microliters were electroporated into E. coli DH12S cells andresulting colonies were screened by PCR, using a primer pair designedfrom the sequence AK124635.1, to identify the proper cDNAs.

Oligos used to identify the cDNA by PCR:

PCSK9-int3s (SEQ ID NO: 8) 5′-CTAGGCCTCCCTTTCCTTGT-3′ PCSK9-int3a (SEQID NO: 9) 5′-TTCCAAGGTGACATTTGTGG-3′

95 colonies were screened, and one cDNA clone was positive by PCR. Thefull-length sequence was obtained.

The full-length nucleotide sequence and the encoded polypeptide forPCSK9b are shown in FIGS. 1A-C.

Analysis of this sequence indicated it was a variant of proproteinconvertase subtilisin/kexin type 9 (PCSK9).

Example 2 Method of Cloning the Novel Human PCSK9c Polynucleotide of thePresent Invention

A method nearly identical to the method described in Example 1 wasutilized to clone the variant PCSK9c with the exceptions that follow.From the sequence NM_174936.2, an anti-sense 80-mer oligo with biotin onthe 5′ end was designed with the following sequence:

(SEQ ID NO: 14) 5′bTGGCAGGCGGCGTTGAGGACGCGGCTGTACCCACCCGCCAGGGGCAGCAGCACCACCAGTGGCCCCACAGGCTGGACCA-3′.and was hybridized to sense single-stranded covalently closed circularbrain/testis cDNA library as described above. Following release from thebiotinylated oligo/streptavidin magnetic bead complex, the cDNA wasprecipitated as described above. The single stranded cDNA was convertedinto double strands as described above except that the following genespecific anti-sense repair primer was used: 5′-GAGTAGAGGCAGGCATCGTC-3′(SEQ ID NO:17). Screening colonies to identify proper cDNAs wasperformed with the following PCR primers:

PCSK9-ex6s (SEQ ID NO: 15) 5′-GCCTGGAGTTTATTCGGAAA-3′ PCSK9-ex6a (SEQ IDNO: 16) 5′-GAGTAGAGGCAGGCATCGTC-3′

95 colonies were screened, and one cDNA clone was positive by PCR. Thefull-length sequence of the cDNA was obtained. This clone was also foundto represent a variant of proprotein convertase subtilisin/kexin type 9(PCSK9).

The full-length nucleotide sequence and the encoded polypeptide forPCSK9c are shown in FIGS. 1A-D.

Example 3 Method of Assessing the Expression Profile of the Novel PCSK9Band PCSK9C Polypeptides of the Present Invention Using Expanded mRNATissue and Cell Sources

Total RNA from tissues was isolated using the TRIZOL® protocol(Invitrogen) and quantified by determining its absorbance at 260 nM. Anassessment of the 18s and 28s ribosomal RNA bands was made by denaturinggel electrophoresis to determine RNA integrity.

The specific sequence to be measured was aligned with related genesfound in GENBANK® to identity regions of significant sequence divergenceto maximize primer and probe specificity. Gene-specific primers andprobes were designed using the ABI primer express software to amplifysmall amplicons (150 base pairs or less) to maximize the likelihood thatthe primers function at 100% efficiency. All primer/probe sequences weresearched against Public Genbank databases to ensure target specificity.Primer and probe sequences were designed to hybridize to regions sharedby both PCSK9b and PCSK9c, in addition to wild type PCSK9. Primers andprobes were obtained from ABI.

For PCSK9b and PCSK9c, the primer probe sequences were as follows:

Forward Primer (SEQ ID NO: 10) 5′-CCTGCGCGTGCTCAACT-3′ Reverse Primer(SEQ ID NO: 11) 5′-CCGAATAAACTCCAGGCCTATG-3′ TAQMAN ® Probe (SEQ ID NO:12) 5′-CCGCTAACCGTGCCCTTCCCTTG-3′

DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA wasdivided into 2 aliquots and one half was treated with Rnase-free Dnase(Invitrogen). Samples from both the Dnase-treated and non-treated werethen subjected to reverse transcription reactions with (RT+) and without(RT−) the presence of reverse transcriptase. TAQMAN® assays were carriedout with gene-specific primers (see above) and the contribution ofgenomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT− non-Dnase treated RNA to thaton the RT+/RT− Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT− RNA must be less that 10% of that obtainedwith Dnased RT+ RNA. If not the RNA was not used in actual experiments.

Reverse Transcription Reaction and Sequence Detection

100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500 μM of each dNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90 C for 5min to denature enzyme.

Quantitative sequence detection was carried out on an ABI PRISM® 7700 byadding to the reverse transcribed reaction 2.5 μM forward and reverseprimers, 2.0 μM of the TAQMAN® probe, 500 μM of each dNTP, buffer and 5U AMPLITAQ GOLD®. The PCR reaction was then held at 94° C. for 12 min,followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

The threshold cycle (Ct) of the lowest expressing tissue (the highest Ctvalue) was used as the baseline of expression and all other tissues wereexpressed as the relative abundance to that tissue by calculating thedifference in Ct value between the baseline and the other tissues andusing it as the exponent in 2^((ΔCt))

The expanded expression profile of the PCSK9b and PCSK9c polypeptides isprovided in FIG. 6, and described elsewhere herein. Table IV identifiesthe analyzed tissues.

TABLE IV Number Tissue 1 adipose_mesenteric_ileum 2blood_vessel_cerebral 3 blood_vessel_pulmonary 4 blood_vessel_renal 5brain_amygdala 6 brain_cerebellum 7 brain_cortex_cingulate_anterior 8brain_cortex_cingulate_posterior 9 brain_cortex_frontal_medial 10brain_cortex_temporal 11 brain_dorsal_raphe_nucleus 12 brain_hippocampus13 brain_hypothalamus_anterior 14 brain_hypothalamus_posterior 15brain_locus_coeruleus 16 brain_medulla_oblongata 17brain_nucleus_accumbens 18 brain_substantia_nigra 19 breast 20 caecum 21colon 22 duodenum 23 heart_left_atria 24 heart_left_ventricle 25 ileum26 jejunum 27 kidney_cortex 28 kidney_medulla 29 kidney_pelvis 30liver_parenchyma 31 lung_bronchus_primary 32 lung_bronchus_tertiary 33lung_parenchyma 34 lymph_gland_tonsil 35 muscle_skeletal 36 oesophagus37 pancreas 38 rectum 39 spinal_cord 40 stomach_antrum 41 stomach_body42 stomach_pyloric_canal 43 testis

Example 4 Method of Characterizing the PCSK9B or PCSK9C Polypeptides ofthe Present Invention

Expression analysis of PCSK9b and PCSK9c polynucleotides andpolypeptides using both quantitative PCR and Western blots, in additionto the affect of PCSK9b and PCSK9c on LDL update by the LDLR, wasassessed according to the methods outlined herein. Briefly:

Materials and Methods

Gene Cloning.

PCSK9 variant b was cloned from a human brain cDNA library, and variantc was clone from a brain/testis cDNA library. Each variant product wasinserted in two expression vectors CD3 MYCHIS and pEF-DESTS1 tagged withHis and V5 or Myc eiptopes at the C-terminus (#3114 for PCSK9 variantb-pCD3 MYCHIS, 3115 for PCSK9 variant b-pEF-DESTS1, #3116 for PCSK9variant c-pCD3 MYCHIS, #3117 for PCSK9 variant c-pEF-DESTS1). Wild typePCSK9 and PCSK9 mutant D374 were cloned by PCR product insertion inpcDNA3 vector.

Cell Culture.

All cells lines including HepG2, Chinese hamster ovary cells (CHO), andHEK293, were derived from American Type Cell Culture (Manassa, Va.).Both HepG2 and HEK293 cells were grown in DMEM medium (Invitrogen Gibco,Carlsbad, Calif.) supplemented with 10% heat inactivated fetal bovineserum (FBS, Gibco), 1 mM sodium pyruvate (Gibco), 2 mM Glutamax (Gibco),100 ug/ml streptomycin sulfate and 100 U/ml penicillin (Gibco). CHOcells were grown with the F12 medium with Glutamax (Gibco) supplementedwith 10% fetal bovine serum.

DiI-LDL Uptake Assay.

15,000 cells per well were plated in the 96-well poly D lysine-coatedplate with the above 10% FBS growth medium and grown in 37° C. incubatorovernight. On day two, the medium was changed to 5% lipoproteindeficient serum (LPDS) growth medium supplemented with 50 μM sodiummevalonate, and 100 ng/ml of an HMG CoA reductase inhibitor. On daythree, aliquots of fluorescent-labeled LDL (DiI-LDL) (source: BiomedicalTechnologies, Stoughton, Mass.) were added into the medium at aconcentration of 10 ug/ml and continued to incubate for two more hours.Formaldehyde was added to the cells at a concentration of 4% togetherwith Hoechst dye at 10 ug/ml. After 20 minute incubation at roomtemperature, cells were washed with PBS three times and read on LJLBiosystems (Molecular Device, Sunnyvale, Calif.) for DNA content at theexcitation wavelength of 360 nm, and emission wavelength of 460 nm.Cells were then lysed by NaOH (0.001 N) and SDS (0.001%). DiI-LDL uptakewas measured on LJL at the excitation wavelength of 540 nm, and emissionwavelength of 580 nm. The DiI-LDL uptake data were normalized to DNAcontent by taking the ratio of DiI-LDL reading to Hoechst dye reading.

mRNA Quantification.

Total RNA isolation from cells was performed by the 6100 Nucleotide AcidPrepStation (ABI, Foster City, Calif.). Cells were lysed by the NucleicAcid Lysis Solution (ABI) following the manufacturer's instruction.Lysed cells were loaded to the instrument for RNA extraction based onthe protocol suggested by the manufacturer. RNA quantity was measured bythe SPECTRAMAX® Plus (Molecular Device, Sunnyvale, Calif.).

Quantitative RT-PCR:

cDNA was prepared by using iScript cDNA synthesis reagents (Biorad,Hercules, and CA) following manufacturer instructions. Quantitative PCRwas performed using iTaq SYBR® Green Supermix with ROX reagents(Bio-Rad) on an ABI PRISM® 7900HT Sequence Detection System. Primersequences for the assays were: wild type PCSK9, forwardTGTCTTGCCCAGAGCATCC (SEQ ID NO:30), reverse TATTCATCCGCCCGGTACC (SEQ IDNO:31); variant b, forward AGATGTCATCAATGAGGCCT (SEQ ID NO:32), reverseAGCTGCCAACCTGCAAAAAC (SEQ ID NO:33); variant b/c, forwardCTCTGAGGTrGTGACTCGTGTGA (SEQ ID NO:34), reverse AGCGTrCTCCACTCCACAAGA(SEQ ID NO:35); LDLR, forward GAGAATGATCTGCAGCACCCA (SEQ ID NO:36),reverse TGCTGATGACGGTGTCATAGG (SEQ ID NO:37). Gene expression levelswere normalized to GAPDH mRNA.

Western Blot Analysis.

Cells were plated in DMEM supplemented with 10% FBS on 6-well plates. Onday two, 4 ug/well of plasmid DNA were transfected by Lipofectamine 2000(Invitrogen) following manufacturer's suggestion. After 24 hr, themedium was switched to DMEM media with 5% LPDS, 50 uM sodium mevalonate,and 100 ng/ml of BMS-423526. After an additional 24 hr, the medium wasremoved and the cells were lysed with the buffer containing 0.1% TritonX-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4) plus Complete ProteaseInhibitor Cocktail (Roche). Protein concentration was assayed withBioRad protein assay (BioRad). 10 ug of proteins from lysate or 15 ulfrom a total of 2 ml conditioned medium were loaded on 4-15% Tris-HClgels (Invitrogen). After protein transfer, membranes were incubated withanti-hPCSK9 primary antibody or anti-LDLR primary antibody, followed byIRdye 800 labeled anti-mouse IgG for PCSK9 or IRdye 680 labeledanti-rabbit IgG for LDLR. Protein bands were subsequently detected usingthe ODYSSEY® Infrared Imaging System (Li-Cor, Lincoln, Nebr.) accordingto manufacturer's instruction.

Results

Overexpression of PCSK9 Variants in Cells.

PCSK9 variant b and c were transfected into HEK and CHO as well as HepG2cells. Both variants were expressed in these cells with mRNA levelscomparable to that of wild type (full length) PCSK9 and thegain-of-function mutant D374Y-PCSK9 (FIG. 7A). As expected, LDLR mRNAlevels were not affected by expression of the PCSK9 mRNAs in these cells(FIG. 7B), consistent with previous studies using known PCSK9 variantsand mutants (Maxwell K. N., Breslow J. L., Proc. Nat. Amer. Sci.,101:7100-7105 (2004); Benjannet S., et al., J. Bio. Chem.279:48865-48875 (2004)). Western blot results using PCSK9 antibodydemonstrated that both variant b and variant c were also expressed asprotein in the transfected cells. Variant b was expressed as anapproximately 33-kDa protein and variant c was expressed as anapproximately 57-kDa protein (FIG. 8A). The apparent molecular weightsof the variant proteins as judged by SDS-PAGE mobility corresponded totheir predicted molecular weight from sequence analysis. Compared withwild type PCSK9 protein, the protein expression levels of the twovariants were lower yet readily detectable by antibody.

In the conditioned media of the cell culture, both PCSK9-b and -cprotein expression were observed by Western blot (FIG. 8B), indicatingthat these two variants may be secreted by the cells.

PCSK9 Variants Exhibit Functional Activity in DiI-LDL Uptake Assay.

DiI-LDL uptake is a standardized functional assay for LDLR receptor,while PCSK9 activity reduces LDLR. Therefore DiI-LDL uptake by cells canbe used as a functional assay for PCSK9 activity. Transient expressionof PCSK9 variant c decreased the uptake of DiI-LDL in HepG2 cells,compared to vector control, indicating that variant c is competent toexpress PCSK9 functional activity on LDLR (FIG. 9A). In this assay,variant c showed greater activity than wild-type PCSK9. Variant b showedgreater activity than wildtype PCSK9, but was less than the PCSK9bvariant. Based on sequence deduction and the molecular weights seen inWestern blots, variant PCSK9c appears to form a nearly complete PCSK9protein (compared to wild type), while variant PCSK9c lacks part of theC-terminal domain, consistent with the activity assay findings.

Consistent with the DiI-LDL uptake assay, expression of PCSK9 variant cdecreased LDLR protein level in CHO cells assayed by Western blot, whileexpression of variant b did not appear to affect LDLR protein levelunder these conditions (FIG. 9B).

Conclusions

The two PCSK9 variants, b and c, were examined in a series ofexperiments to detect expression and function These two variants differfrom the wild type PCSK9 by the lack of the signal peptide and prodomainas well as 22 amino acid in the N-terminus of the catalytic domain. Invariant b, a portion of the C-terminal domain is also absent.

Both variants were expressed at the mRNA level in basal (sterolsuppressed) HepG2 cells, with mRNA approximately 1% of the wild-typePCSK9 mRNA level in the presence of medium containing 10% fetal bovineserum (data not shown). Media containing 10% fetal bovine serum (FBS) isknown to suppress sterol-sensitive genes, including PCSK9. However,under conditions including 5% lipoprotein-deficient serum (LPDS) in themedium, which is known to induce sterol-sensitive genes including PCSK9,the PCSK9c variant mRNA was highly induced, with levels reachingapproximately 10% of the wild-type PCSK9 mRNA level (data not shown).Transient expression of the two variants using standard plasmidexpression vectors in mammalian cell lines resulted in the PCSK9bvariant being expressed as a 33 kDa protein, and the PCSK9c variantbeing expressed as a 57 kDa protein. Both of the PCSK9 variant proteinswere detectable in conditioned medium as well as in cell lysates,indicating that both of them may also be secreted from cells. Based onthe DiI-LDL uptake assay and western blot data, transient expression ofvariant c decreased LDLR levels in cells, while variant b exhibitedlesser effects.

These data suggest that the novel PCSK9 variants, especially variant c,are not only expressed but are functionally competent and exhibit PCSK9activity by interacting with the LDLR and decreasing LDLR levels, themain function identified for wild-type PCSK9 to date. The data support abiological role for the novel variants in modulating the LDLR andsuggest they are useful in biotechnology and discovery applicationsinvolving PCSK9 expression, function, and screening.

Example 5 Method of Measuring the Proteinase Activity of the PCSK9B orPCSK9C Polypeptides of the Present Invention

A number of assays known in the art may be utilized to demonstrate theproteinase activity of the PCSK9b and PCSK9c polypeptides of the presentinvention. Specifically, the method outlined in Naureckiene et al.(Arch. Biochem. Biophys., 420:55-67 (2003); which is hereby incorporatedby reference in its entirety herein) may be utilized. Briefly:

Expression of PCSK9b and PCSK9c

Escherichia coli BL21 (DE3) cells may be transformed withprN1(ΔSP;ΔC@Q453)/6×His. Transformants are grown in LB medium(supplemented to 100 μg/ml with carbenicillin) at 30° C. to an OD₆₀₀ of0.6. Expression is induced with 0.1 mM IPTG and the culture grown for anadditional 3.5 h. Cells are harvested by centrifugation 30 min. at14,000 g, and stored at −80 C.

Purification of PCSK9b and PCSK9c

Procedures may be performed at 4° C. Cells from 2 L of culture areresuspended in 25 ml lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl;10% glycerol; and 1 M NDSB) in the presence or absence of an EDTA-freeproteinase inhibitor cocktail (Roche). After cell lysis by sonication,the cell debris is removed by centrifugation (30 min, 14,000 g). Thesupernatant containing soluble proteins is loaded onto apre-equilibrated 1 ml Ni-nitrilotriacetic acid (NTA) Agarose column(Qiagen). The column is washed with 20 ml lysis buffer containing 20 mMimidazole to remove weakly bound contaminating proteins. The purifiedprotein is eluted with 5 ml lysis buffer containing 200 mM imidazole.Eluted protein is dialyzed overnight against 1 L Buffer A: 50 mMTris-HCl, pH 8.0, 50 mM NaCl, and 10% glycerol. Dialyzed protein isloaded onto a 1 ml MONO Q® column (Amersham Biosciences) equilibratedwith the same buffer. The column is washed with 10 ml of the same bufferand proteins are eluted with 12 ml NaCl gradient (0.05-1.0 M) in thesame buffer. One milliliter fractions are collected and analyzed forpurity on SDS-PAGE. Fractions containing purified protein are pooled anddialyzed against 1000 ml storage buffer (50 mM Tris-HCl, pH 8.0, 50 mMNaCl, and 10% glycerol), aliquoted, and stored at −80 C. In parallel, apurification from cells transformed with empty vector pET21a isperformed to generate material for use as a negative control inenzymatic assays.

Proteinase Activity Assay

Subtilisin-, furin-, and TPP-specific fluorogenic substrates may bepurchased from Bachem. Custom-made proteinase substrates can besynthesized by New England Peptide, Inc. Sequences of peptide substratesare as follows:

Peptide Sequence A Dnp-FAQSIPK-AMC (SEQ ID NO: 18) B Dnp-DSLVFAK-AMC(SEQ ID NO: 19) C Dnp-FANAIPK-AMC (SEQ ID NO: 20)

Proteolytic activity may be assayed at 37° C. using 100 μM substrate and0.12 M enzyme in various buffers, including 50 mM Tris-HCL, pH 7.5 to pH11, 1 mM to 20 mM CaCl2, and 0.5% TX-100 at 37° C. Protein derived froman empty vector transformation is included as a negative control forenzymatic activity. Continuous and end-point fluorimetric measurementsmay be performed on spectrophotometer (λ_(ex)=0.350 nm, λ_(em)=450 nm).

Example 6 Method of Screening for Compounds that Interact with thePCSK9B or PCSK9C Polypeptide

The following assays are designed to identify compounds that bind to thePCSK9b or PCSK9c polypeptide, bind to other cellular proteins thatinteract with the PCSK9b or PCSK9c polypeptide, and to compounds thatinterfere with the interaction of the PCSK9b or PCSK9c polypeptide withother cellular proteins.

Such compounds can include, but are not limited to, other cellularproteins. Specifically, such compounds can include, but are not limitedto, peptides, such as, for example, soluble peptides, including, but notlimited to Ig-tailed fusion peptides, comprising extracellular portionsof PCSK9b or PCSK9c polypeptide transmembrane receptors, and members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghton, R. et al., 1991, Nature 354:84-86), made of D-and/or L-configuration amino acids, phosphopeptides (including, but notlimited to, members of random or partially degenerate phosphopeptidelibraries; see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′).sub.2 and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can beuseful, for example, in elaborating the biological function of thePCSK9b or PCSK9c polypeptide, and for ameliorating symptoms of tumorprogression, for example. In instances, for example, whereby a tumorprogression state or disorder results from a lower overall level ofPCSK9b or PCSK9c expression, PCSK9b or PCSK9c polypeptide, and/or PCSK9bor PCSK9c polypeptide activity in a cell involved in the tumorprogression state or disorder, compounds that interact with the PCSK9bor PCSK9c polypeptide can include ones which accentuate or amplify theactivity of the bound PCSK9b or PCSK9c polypeptide. Such compounds wouldbring about an effective increase in the level of PCSK9b or PCSK9cpolypeptide activity, thus ameliorating symptoms of the tumorprogression disorder or state. In instances whereby mutations within thePCSK9b or PCSK9c polypeptide cause aberrant PCSK9b or PCSK9cpolypeptides to be made which have a deleterious effect that leads totumor progression, compounds that bind PCSK9b or PCSK9c polypeptide canbe identified that inhibit the activity of the bound PCSK9b or PCSK9cpolypeptide. Assays for testing the effectiveness of such compounds areknown in the art and discussed, elsewhere herein.

Example 7 Method of Identifying Compounds that Interfere with PCSK9B orPCSK9C Polypeptide/Cellular Product Interaction

The PCSK9b or PCSK9c polypeptide of the invention can, in vivo, interactwith one or more cellular or extracellular macromolecules, such asproteins. Such macromolecules include, but are not limited to,polypeptides, particularly PCSK9 ligands, and those products identifiedvia screening methods described, elsewhere herein. For the purposes ofthis discussion, such cellular and extracellular macromolecules arereferred to herein as “binding partner(s)”. For the purpose of thepresent invention, “binding partner” may also encompass polypeptides,small molecule compounds, polysaccarides, lipids, and any other moleculeor molecule type referenced herein. Compounds that disrupt suchinteractions can be useful in regulating the activity of the PCSK9b orPCSK9c polypeptide, especially mutant PCSK9b or PCSK9c polypeptide. Suchcompounds can include, but are not limited to molecules such asantibodies, peptides, and the like described in elsewhere herein.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the PCSK9b or PCSK9c polypeptideand its cellular or extracellular binding partner or partners involvespreparing a reaction mixture containing the PCSK9b or PCSK9cpolypeptide, and the binding partner under conditions and for a timesufficient to allow the two products to interact and bind, thus forminga complex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of PCSK9bor PCSK9c polypeptide and its cellular or extracellular binding partner.Control reaction mixtures are incubated without the test compound orwith a placebo. The formation of any complexes between the PCSK9b orPCSK9c polypeptide and the cellular or extracellular binding partner isthen detected. The formation of a complex in the control reaction, butnot in the reaction mixture containing the test compound, indicates thatthe compound interferes with the interaction of the PCSK9b or PCSK9cpolypeptide and the interactive binding partner. Additionally, complexformation within reaction mixtures containing the test compound andnormal PCSK9b or PCSK9c polypeptide can also be compared to complexformation within reaction mixtures containing the test compound andmutant PCSK9b or PCSK9c polypeptide. This comparison can be important inthose cases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal PCSK9b or PCSK9c polypeptide.

The assay for compounds that interfere with the interaction of thePCSK9b or PCSK9c polypeptide and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the PCSK9b or PCSK9c polypeptide or the binding partneronto a solid phase and detecting complexes anchored on the solid phaseat the end of the reaction. In homogeneous assays, the entire reactionis carried out in a liquid phase. In either approach, the order ofaddition of reactants can be varied to obtain different informationabout the compounds being tested. For example, test compounds thatinterfere with the interaction between the PCSK9b or PCSK9c polypeptideand the binding partners, e.g., by competition, can be identified byconducting the reaction in the presence of the test substance; i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with the PCSK9b or PCSK9c polypeptide and interactivecellular or extracellular binding partner. Alternatively, test compoundsthat disrupt preformed complexes, e.g. compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

In a heterogeneous assay system, either the PCSK9b or PCSK9c polypeptideor the interactive cellular or extracellular binding partner, isanchored onto a solid surface, while the non-anchored species islabeled, either directly or indirectly. In practice, microtitre platesare conveniently utilized. The anchored species can be immobilized bynon-covalent or covalent attachments. Non-covalent attachment can beaccomplished simply by coating the solid surface with a solution of thePCSK9b or PCSK9c polypeptide or binding partner and drying.Alternatively, an immobilized antibody specific for the species to beanchored can be used to anchor the species to the solid surface. Thesurfaces can be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the PCSK9b or PCSK9cpolypeptide and the interactive cellular or extracellular bindingpartner product is prepared in which either the PCSK9b or PCSK9cpolypeptide or their binding partners are labeled, but the signalgenerated by the label is quenched due to complex formation (see, e.g.,U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances which disrupt PCSK9b or PCSK9c polypeptide-cellular orextracellular binding partner interaction can be identified.

In a particular embodiment, the PCSK9b or PCSK9c polypeptide can beprepared for immobilization using recombinant DNA techniques known inthe art. For example, the PCSK9b or PCSK9c polypeptide coding region canbe fused to a glutathione-S-transferase (GST) gene using a fusion vectorsuch as pGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion product. The interactive cellular orextracellular product can be purified and used to raise a monoclonalantibody, using methods routinely practiced in the art and describedabove. This antibody can be labeled with the radioactive isotope.sup.125 I, for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-PCSK9b or PCSK9c polypeptide fusionproduct can be anchored to glutathione-agarose beads. The interactivecellular or extracellular binding partner product can then be added inthe presence or absence of the test compound in a manner that allowsinteraction and binding to occur. At the end of the reaction period,unbound material can be washed away, and the labeled monoclonal antibodycan be added to the system and allowed to bind to the complexedcomponents. The interaction between the PCSK9b or PCSK9c polypeptide andthe interactive cellular or extracellular binding partner can bedetected by measuring the amount of radioactivity that remainsassociated with the glutathione-agarose beads. A successful inhibitionof the interaction by the test compound will result in a decrease inmeasured radioactivity.

Alternatively, the GST-PCSK9b or PCSK9c polypeptide fusion product andthe interactive cellular or extracellular binding partner product can bemixed together in liquid in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the bindingpartners are allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the PCSK9b or PCSK9c polypeptide product and the interactive cellularor extracellular binding partner (in case where the binding partner is aproduct), in place of one or both of the full length products.

Any number of methods routinely practiced in the art can be used toidentify and isolate the protein's binding site. These methods include,but are not limited to, mutagenesis of one of the genes encoding one ofthe products and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can be selected. Sequenceanalysis of the genes encoding the respective products will reveal themutations that correspond to the region of the product involved ininteractive binding. Alternatively, one product can be anchored to asolid surface using methods described in this Section above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain can remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the cellular orextracellular binding partner product is obtained, short gene segmentscan be engineered to express peptide fragments of the product, which canthen be tested for binding activity and purified or synthesized.

Example 8 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC® Deposit Numbercited in Table I for any given cDNA clone also may contain one or moreadditional plasmids, each comprising a cDNA clone different from thatgiven clone. Thus, deposits sharing the same ATCC® Deposit Numbercontain at least a plasmid for each cDNA clone identified in Table I.Typically, each ATCC® deposit sample cited in Table I comprises amixture of approximately equal amounts (by weight) of about 1-10 plasmidDNAs, each containing a different cDNA clone and/or partial cDNA clone;but such a deposit sample may include plasmids for more or less than 2cDNA clones.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table I.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO: 1.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-(-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:1 or SEQ ID NO:3 (i.e., within the region of SEQ IDNO:1 or SEQ ID NO:3 bounded by the 5′ NT and the 3′ NT of the clonedefined in Table I) are synthesized and used to amplify the desired cDNAusing the deposited cDNA plasmid as a template. The polymerase chainreaction is carried out under routine conditions, for instance, in 25 ulof reaction mixture with 0.5 ug of the above cDNA template. A convenientreaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each ofdATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taqpolymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C.for 1 min) are performed with a Perkin-Elmer Cetus automated thermalcycler. The amplified product is analyzed by agarose gel electrophoresisand the DNA band with expected molecular weight is excised and purified.The PCR product is verified to be the selected sequence by subcloningand sequencing the DNA product.

The polynucleotide(s) of the present invention, the polynucleotideencoding the polypeptide of the present invention, or the polypeptideencoded by the deposited clone may represent partial, or incompleteversions of the complete coding region (i.e., full-length gene). Severalmethods are known in the art for the identification of the 5′ or 3′non-coding and/or coding portions of a gene which may not be present inthe deposited clone. The methods that follow are exemplary and shouldnot be construed as limiting the scope of the invention. These methodsinclude but are not limited to, filter probing, clone enrichment usingspecific probes, and protocols similar or identical to 5′ and 3′ “RACE”protocols that are well known in the art. For instance, a method similarto 5′ RACE is available for generating the missing 5′ end of a desiredfull-length transcript. (Fromont-Racine et al., Nucleic Acids Res.21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA that may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988).Briefly, a cDNA clone missing either the 5′ or 3′ end can bereconstructed to include the absent base pairs extending to thetranslational start or stop codon, respectively. In some cases, cDNAsare missing the start of translation, therefor. The following brieflydescribes a modification of this original 5′ RACE procedure. Poly A+ ortotal RNAs reverse transcribed with SUPERSCRIPT II (Gibco/BRL) and anantisense or I complementary primer specific to the cDNA sequence. Theprimer is removed from the reaction with a MICROCON® Concentrator(Amicon). The first-strand cDNA is then tailed with dATP and terminaldeoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence isproduced which is needed for PCR amplification. The second strand issynthesized from the dA-tail in PCR buffer, Taq DNA polymerase(Perkin-Elmer Cetus), an oligo-dT primer containing three adjacentrestriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primercontaining just these restriction sites. This double-stranded cDNA isPCR amplified for 40 cycles with the same primers as well as a nestedcDNA-specific antisense primer. The PCR products are size-separated onan ethidium bromide-agarose gel and the region of gel containing cDNAproducts the predicted size of missing protein-coding DNA is removed.cDNA is purified from the agarose with the Magic PCR Prep kit (Promega),restriction digested with XhoI or SalI, and ligated to a plasmid such asPBLUESCRIPT® SKII (Stratagene) at XhoI and EcoRV sites. This DNA istransformed into bacteria and the plasmid clones sequenced to identifythe correct protein-coding inserts. Correct 5′ ends are confirmed bycomparing this sequence with the putatively identified homologue andoverlap with the partial cDNA clone. Similar methods known in the artand/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase.Similar reagents and methods to those above are supplied in kit formfrom Gibco/BRL for both 5′ and 3′ RACE for recovery of full lengthgenes. A second kit is available from Clontech which is a modificationof a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32 (1991). The major differences in procedure are that the RNAis alkaline hydrolyzed after reverse transcription and RNA ligase isused to join a restriction site-containing anchor primer to thefirst-strand cDNA. This obviates the necessity for the dA-tailingreaction which results in a polyT stretch that is difficult to sequencepast.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNAlibrary double-stranded DNA. An asymmetric PCR-amplified antisense cDNAstrand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to ObtainFull Length Genes

Once a gene of interest is identified, several methods are available forthe identification of the 5′ or 3′ portions of the gene which may not bepresent in the original cDNA plasmid. These methods include, but are notlimited to, filter probing, clone enrichment using specific probes andprotocols similar and identical to 5′ and 3′RACE. While the full-lengthgene may be present in the library and can be identified by probing, auseful method for generating the 5′ or 3′ end is to use the existingsequence information from the original cDNA to generate the missinginformation. A method similar to 5′RACE is available for generating themissing 5′ end of a desired full-length gene. (This method was publishedby Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)).Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably 30 containing full-length gene RNAtranscript and a primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest, is used to PCR amplify the 5′ portion of the desiredfull length gene which may then be sequenced and used to generate thefull length gene. This method starts with total RNA isolated from thedesired source, poly A RNA may be used but is not a prerequisite forthis procedure. The RNA preparation may then be treated with phosphataseif necessary to eliminate 5′ phosphate groups on degraded or damaged RNAwhich may interfere with the later RNA ligase step. The phosphatase ifused is then inactivated and the RNA is treated with tobacco acidpyrophosphatase in order to remove the cap structure present at the 5′ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the5′ end of the cap cleaved RNA which can then be ligated to an RNAoligonucleotide using T4 RNA ligase. This modified RNA preparation canthen be used as a template for first strand cDNA synthesis using a genespecific oligonucleotide. The first strand synthesis reaction can thenbe used as a template for PCR amplification of the desired 5′ end usinga primer specific to the ligated RNA oligonucleotide and a primerspecific to the known sequence of the apoptosis related of interest. Theresultant product is then sequenced and analyzed to confirm that the 5′end sequence belongs to the relevant apoptosis related.

Example 9 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined herein, to synthesize insertionfragments. The primers used to amplify the cDNA insert should preferablycontain restriction sites, such as BamHI and XbaI, at the 5′ end of theprimers in order to clone the amplified product into the expressionvector. For example, BamHI and XbaI correspond to the restriction enzymesites on the bacterial expression vector pQE-9. (Qiagen, Inc.,Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance(Ampr), a bacterial origin of replication (ori), an IPTG-regulatablepromoter/operator (P/O), a ribosome binding site (RBS), a 6-histidinetag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, that expresses the lacI repressorand also confers kanamycin resistance (Kanr). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6 M-1 M urea gradient in 500 mMNaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing proteaseinhibitors. The renaturation should be performed over a period of 1.5hours or more. After renaturation the proteins are eluted by theaddition of 250 mM imidazole. Imidazole is removed by a final dialyzingstep against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl.The purified protein is stored at 4 degree C. or frozen at −80 degree C.

Example 10 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E. coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., POROS® HS-50, Perceptive Biosystems). The columnis washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner.The absorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (POROS® HQ-50, PerceptiveBiosystems) and weak anion (POROS® CM-20, Perceptive Biosystems)exchange resins. The columns are equilibrated with 40 mM sodium acetate,pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200mM NaCl. The CM-20 column is then eluted using a 10 column volume lineargradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 MNaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected underconstant A280 monitoring of the effluent. Fractions containing thepolypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Coomassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 11 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pAc373 is used to insert apolynucleotide into a baculovirus to express a polypeptide. A typicalbaculovirus expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby convenient restriction sites, which may include, for example BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is often used for efficient polyadenylation. For easy selectionof recombinant virus, the plasmid contains the beta-galactosidase genefrom E. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pVL941 and pAcIM1, as one skilled in the art would readilyappreciate, as long as the construct provides appropriately locatedsignals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in herein, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites at the 5′ end of the primers inorder to clone the amplified product into the expression vector.Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified elsewhere herein (if applicable), is amplified usingthe PCR protocol described herein. If the naturally occurring signalsequence is used to produce the protein, the vector used does not need asecond signal peptide. Alternatively, the vector can be modified toinclude a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (GENECLEAN®, BIO 101 Inc., La Jolla, Ca.).The fragment then is digested with appropriate restriction enzymes andagain purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (GENECLEAN®, BIO 101Inc., La Jolla, Ca.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transformedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BACULOGOLD® baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BACULOGOLD® virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul LIPOFECTIN® plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC®CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of35S-methionine and 5 uCi 35S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 12 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC®37152), pSV2dhfr (ATCC® 37146), pBC12MI (ATCC®67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

The transformed gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991).) Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

A polynucleotide of the present invention is amplified according to theprotocol outlined in herein. If the naturally occurring signal sequenceis used to produce the protein, the vector does not need a second signalpeptide. Alternatively, if the naturally occurring signal sequence isnot used, the vector can be modified to include a heterologous signalsequence. (See, e.g., WO 96/34891.) The amplified fragment is isolatedfrom a 1% agarose gel using a commercially available kit (GENECLEAN®,BIO 101 Inc., La Jolla, Ca.). The fragment then is digested withappropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransformation. Five μg of an expression plasmid is cotransformed with0.5 ug of the plasmid pSVneo using LIPOFECTIN® (Felgner et al., supra).The plasmid pSV2-neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 13 Method of Creating N- and C-Terminal Deletion MutantsCorresponding to PCSK9, PCSK9B or PCSK9C Polypeptides of the PresentInvention

As described elsewhere herein, the present invention encompasses thecreation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to thePCSK9, PCSK9b or PCSK9c polypeptide of the present invention. A numberof methods are available to one skilled in the art for creating suchmutants. Such methods may include a combination of PCR amplification andgene cloning methodology. Although one of skill in the art of molecularbiology, through the use of the teachings provided or referenced herein,and/or otherwise known in the art as standard methods, could readilycreate each deletion mutant of the present invention, exemplary methodsare described below.

Briefly, using the isolated cDNA clone encoding the full-length PCSK9bor PCSK9c polypeptide sequence (as described herein, for example),appropriate primers of about 15-25 nucleotides derived from the desired5′ and 3′ positions of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:38 may bedesigned to PCR amplify, and subsequently clone, the intended N- and/orC-terminal deletion mutant. Such primers could comprise, for example, aninititation and stop codon for the 5′ and 3′ primer, respectively. Suchprimers may also comprise restriction sites to facilitate cloning of thedeletion mutant post amplification. Moreover, the primers may compriseadditional sequences, such as, for example, flag-tag sequences, kozacsequences, or other sequences discussed and/or referenced herein.

For example, in the case of the PCSK9b L16 to R315 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

5′ Primer (SEQ ID NO: 22) 5′-GCAGCAGCGGCCGC CTAGACACCAGCATACAGAGTGACC-3′            NotI 3′ Primer (SEQ IDNO: 23) 5′-GCAGCA GTCGAC TCTGGGGCGCAGCGGGCGATGGCTG-3′           SalI

For example, in the case of the PCSK9b M1 to P284 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

5′ Primer (SEQ ID NO: 24) 5′-GCAGCAGCGGCCGC ATGTCGCCTTGGAAAGACGGAGGCA-3′            NotI 3′ Primer (SEQ IDNO: 25) 5′-GCAGCA GTCGAC AGGGCCTGCCCCATGGGTGCTGGGG-3′           SalI

For example, in the case of the PCSK9c L16 to Q523 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

5′ Primer (SEQ ID NO: 26) 5′-GCAGCAGCGGCCGC CTAGACACCAGCATACAGAGTGACC-3′            NotI 3′ Primer (SEQ IDNO: 27) 5′-GCAGCA GTCGAC CTGGAGCTCCTGGGAGGCCTGCGCC-3′           SalI

For example, in the case of the PCSK9c M1 to A306 C-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

5′ Primer (SEQ ID NO: 28) 5′-GCAGCAGCGGCCGC ATGTCGCCTTGGAAAGACGGAGGCA-3′            NotI 3′ Primer (SEQ IDNO: 29) 5′-GCAGCA GTCGAC GGCGATGGCTGTGGCCATCCGTGTA-3′           SalI

For example, in the case of the PCSK9 L16 to Q692 N-terminal deletionmutant, the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant:

5′ Primer (SEQ ID NO: 39) 5′-GCAGCAGCGGCCGC CTGCTGCTGCTGCTGCTGCTGCTCC-3′            NotI 3′ Primer (SEQ IDNO: 40) 5′-GCAGCA GTCGAC CTGGAGCTCCTGGGAGGCCTGCGCC-3′           SalI

Representative PCR amplification conditions are provided below, althoughthe skilled artisan would appreciate that other conditions may berequired for efficient amplification. A 100 ul PCR reaction mixture maybe prepared using 10 ng of the template DNA (cDNA clone of PCSK9b orPCSK9c), 200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNA polymerase (PE),and standard Taq DNA polymerase buffer. Typical PCR cycling conditionare as follows:

-   -   20-25 cycles: 45 sec, 93 degrees        -   2 min, 50 degrees        -   2 min, 72 degrees    -   1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5 U Klenow Fragment may be addedand incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of PCSK9b (SEQ ID NO:1) or PCSK9c(SEQ ID NO:3) or PCSK9 (SEQ ID NO:38), and ‘X’ is equal to the mostN-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 5′primer, while the second term will provide the end 3′ nucleotideposition of the 5′ primer corresponding to sense strand of SEQ ID NO:1or SEQ ID NO:3 or SEQ ID NO:38. Once the corresponding nucleotidepositions of the primer are determined, the final nucleotide sequencemay be created by the addition of applicable restriction site sequencesto the 5′ end of the sequence, for example. As referenced herein, theaddition of other sequences to the 5′ primer may be desired in certaincircumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:(S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of PCSK9b (SEQ ID NO:1) or PCSK9c(SEQ ID NO:3) or PCSK9 (SEQ ID NO:38), and ‘X’ is equal to the mostC-terminal amino acid of the intended N-terminal deletion mutant. Thefirst term will provide the start 5′ nucleotide position of the 3′primer, while the second term will provide the end 3′ nucleotideposition of the 3′ primer corresponding to the anti-sense strand of SEQID NO:1 or SEQ ID NO:3 or SEQ ID NO:38. Once the correspondingnucleotide positions of the primer are determined, the final nucleotidesequence may be created by the addition of applicable restriction sitesequences to the 5′ end of the sequence, for example. As referencedherein, the addition of other sequences to the 3′ primer may be desiredin certain circumstances (e.g., stop codon sequences, etc.). The skilledartisan would appreciate that modifications of the above nucleotidepositions may be necessary for optimizing PCR amplification.

The same general formulas provided above may be used in identifying the5′ and 3′ primer sequences for amplifying any C-terminal deletion mutantof the present invention. Moreover, the same general formulas providedabove may be used in identifying the 5′ and 3′ primer sequences foramplifying any combination of N-terminal and C-terminal deletion mutantof the present invention. The skilled artisan would appreciate thatmodifications of the above nucleotide positions may be necessary foroptimizing PCR amplification.

Example 14 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (as described herein; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the half-life time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector. Note that the polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce theprotein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.)

Human IgG Fc Region

(SEQ ID NO: 21) GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 15 Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing a polypeptide of the present invention areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of theprotein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal antibodies can be prepared using hybridoma technology. (Khleret al., Nature 256:495 (1975); Khler et al., Eur. J. Immunol. 6:511(1976); Khler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.563-681 (1981).) In general, such procedures involve immunizing ananimal (preferably a mouse) with polypeptide or, more preferably, with apolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C.), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC®.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodythat binds to a second antibody. In accordance with this method, proteinspecific antibodies are used to immunize an animal, preferably a mouse.The splenocytes of such an animal are then used to produce hybridomacells, and the hybridoma cells are screened to identify clones thatproduce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab)2 fragments). Alternatively, protein-bindingfragments can be produced through the application of recombinant DNAtechnology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

Moreover, in another preferred method, the antibodies directed againstthe polypeptides of the present invention may be produced in plants.Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

Example 16 Regulation of Protein Expression Via Controlled Aggregationin the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreigngenes, either under the control of an inducible, constitutively active,or endogenous promoter, into organisms. Of particular interest are theinducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci.USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc.), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of aprotein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830 (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences ofthe present invention with one or more copies (preferably at least 2, 3,4, or more) of a conditional aggregation domain (CAD) a domain thatinteracts with itself in a ligand-reversible manner (i.e., in thepresence of an inducing agent) using molecular biology methods known inthe art and discussed elsewhere herein. The CAD domain may be the mutantdomain isolated from the human FKBP 12 (Phe³⁶ to Met) protein (asdisclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)x domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

The specific methods described herein for controlling protein secretionlevels through controlled ER aggregation are not meant to be limitingare would be generally applicable to any of the polynucleotides andpolypeptides of the present invention, including variants, homologues,orthologs, and fragments therein.

Example 17 Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

Many eukaryotic cell surface and proteins are post-translationallyprocessed to incorporate N-linked and O-linked carbohydrates (Kornfeldand Kornfeld (1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al.,(1988) Annu. Rev. Biochem. 57:785-838). Protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994)Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473;Caton et al., (1982) Cell, 37:417-427; Alexamnder and Elder (1984),Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem.,269:14015-14020). In higher organisms, the nature and extent ofglycosylation can markedly affect the circulating half-life andbio-availability of proteins by mechanisms involving receptor mediateduptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol.,41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54).Receptor systems have been identified that are thought to play a majorrole in the clearance of serum proteins through recognition of variouscarbohydrate structures on the glycoproteins (Stockert (1995), Physiol.Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys.,298:49-55). Thus, production strategies resulting in incompleteattachment of terminal sialic acid residues might provide a means ofshortening the bioavailability and half-life of glycoproteins.Conversely, expression strategies resulting in saturation of terminalsialic acid attachment sites might lengthen protein bioavailability andhalf-life.

In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992),J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). Whileglycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in onespecies will be recognized by another species glycosylation machinery.Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E. coli, yeast, orviral organisms; or an E. coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computeralgorithms capable of predicting the location of glycosylation siteswithin a protein. For example, the Motif computer program (GeneticsComputer Group suite of programs) provides this function, as well.

Example 18 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

Although many of the most biologically active proteins known are highlyeffective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

Thus, one aspect of the present invention relates to the ability toenhance specific characteristics of invention through directed molecularevolution. Such an enhancement may, in a non-limiting example, benefitthe inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

Directed evolution is comprised of several steps. The first step is toestablish a library of variants for the gene or protein of interest. Themost important step is to then select for those variants that entail theactivity you wish to identify. The design of the screen is essentialsince your screen should be selective enough to eliminate non-usefulvariants, but not so stringent as to eliminate all variants. The laststep is then to repeat the above steps using the best variant from theprevious screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

Random mutagenesis has been the most widely recognized method to date.Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

While both of the aforementioned methods are effective for creatingrandomized pools of macromolecule variants, a third method, termed “DNAShuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) hasrecently been elucidated. DNA shuffling has also been referred to as“directed molecular evolution”, “exon-shuffling”, “directed enzymeevolution”, “in vitro evolution”, and “artificial evolution”. Suchreference terms are known in the art and are encompassed by theinvention. This new, preferred, method apparently overcomes thelimitations of the previous methods in that it not only propagatespositive traits, but simultaneously eliminates negative traits in theresulting progeny.

DNA shuffling accomplishes this task by combining the principal of invitro recombination, along with the method of “error-prone” PCR. Ineffect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments-further diversifying the potential hybridization sitesduring the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNAshuffling reaction. However, specific reaction conditions for DNAshuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction.Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

Once the DNA substrate has been purified, it would be subjected to DnaseI digestion. About 2-4 ug of the DNA substrate(s) would be digested with0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resultingfragments of 10-50 bp could then be purified by running them through a2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using MICROCON®concentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45cycles, followed by 72 C for 5 min using an MJ RESEARCH® (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s,and 72 C for 30 s). The referred primers would be primers correspondingto the nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purifiedusing methods well known in the art (e.g., Qiagen PCR purification kits)and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published todate, such variations would be obvious to the skilled artisan and areencompassed by the invention. The DNA shuffling method can also betailored to the desired level of mutagenesis using the methods describedby Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it canthen be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficialmutations. When combined with screening, DNA shuffling allows thediscovery of the best mutational combinations and does not assume thatthe best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

Finally, recombination enables parallel processing. This represents asignificant advantage since there are likely multiple characteristicsthat would make a protein more desirable (e.g. solubility, activity,etc.). Since it is increasingly difficult to screen for more than onedesirable trait at a time, other methods of molecular evolution tend tobe inhibitory. However, using recombination, it would be possible tocombine the randomized fragments of the best representative variants forthe various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

Likewise, the invention encompasses the application of DNA shufflingtechnology to the evolution of polynucleotides and polypeptides of theinvention, wherein one or more cycles of DNA shuffling include, inaddition to the gene template DNA, oligonucleotides coding for knownallelic sequences, optimized codon sequences, known variant sequences,known polynucleotide polymorphism sequences, known ortholog sequences,known homologue sequences, additional homologous sequences, additionalnon-homologous sequences, sequences from another species, and any numberand combination of the above.

In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 19 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., usingbuffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

PCR products are cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to the methods described herein are nick-translatedwith digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), andFISH performed as described in Johnson et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISEE® GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 20 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described elsewhere herein. The wells are blockedso that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 21 Method of Isolating Antibody Fragments Directed AgainstPCSK9b or PCSK9c from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstPCSK9b or PCSK9c to which the donor may or may not have been exposed(see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference inits entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37 Cfor 45 minutes without shaking and then at 37 C for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37 C withshaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 370 C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37 C and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37 C. TheE. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Moreover, in another preferred method, the antibodies directed againstthe polypeptides of the present invention may be produced in plants.Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

What is claimed is:
 1. An isolated antibody or antigen binding fragmentthereof that binds to a polypeptide comprising SEQ ID NO:2.
 2. Theisolated antibody of claim 1, wherein said antibody is a monoclonalantibody.
 3. The isolated antibody of claim 1, wherein said antibody isa polyclonal antibody.
 4. A pharmaceutical composition comprising atherapeutically effective amount of the antibody or antigen bindingfragment of claim 1 and a pharmaceutically acceptable carrier.
 5. Anisolated antibody or antigen binding fragment thereof that binds to apolypeptide comprising SEQ ID NO:4.
 6. The isolated antibody of claim 5,wherein said antibody is a monoclonal antibody.
 7. The isolated antibodyof claim 5, wherein said antibody is a polyclonal antibody.
 8. Apharmaceutical composition comprising a therapeutically effective amountof the antibody or antigen binding fragment of claim 5 and apharmaceutically acceptable carrier.